Urinary bladder smooth muscle ion channels: expression, function, and regulation in health and disease

Abstract

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and ultimately determines the two main attributes of the organ: urine storage and voiding. The two functions are facilitated by UBSM relaxation and contraction, respectively, which depend on UBSM excitability shaped by multiple ion channels. In this review, we summarize the current understanding of key ion channels establishing and regulating UBSM excitability and contractility. They include excitation-enhancing voltage-gated Ca²⁺ (Ca_v) and transient receptor potential channels, excitation-reducing K⁺ channels, and still poorly understood Cl⁻ channels. Dynamic interplay among UBSM ion channels determines the overall level of Ca_v channel activity. The net Ca²⁺ influx via Ca_v channels increases global intracellular Ca²⁺ concentration, which subsequently triggers UBSM contractility. Here, for each ion channel type, we describe UBSM tissue/cell expression (mRNA and protein) profiles and their role in regulating excitability and contractility of UBSM in various animal species, including the mouse, rat, and guinea pig, and, most importantly, humans. The currently available data reveal certain interspecies differences, which complicate the translational value of published animal research results to humans. This review highlights recent developments, findings on genetic knockout models, pharmacological data, reports on UBSM ion channel dysfunction in animal bladder disease models, and the very limited human studies currently available. Among all gaps in present-day knowledge, the unknowns on expression and functional roles for ion channels determined directly in human UBSM tissues and cells under both normal and disease conditions remain key hurdles in the field.

INTRODUCTION

Urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, forms the bladder wall and determines the two main functions of the organ: urine storage and voiding (9). UBSM relaxation facilitates storage, while contraction coupled with the opening of the urinary bladder sphincter aids in voiding. Structurally, UBSM cells are organized into muscle bundles. Within each bundle, UBSM cells form a well-coupled electrical syncytium. In contrast, the coupling among distinct adjacent smooth muscle bundles is weak (9, 19). Collagen fibers, connective tissue, nerve processes and varicosities, and other cells, including recently characterized platelet-derived growth factor receptor-α (PDGFRα)⁺ interstitial cells, populate the space between UBSM fibers and determine bladder function via functional interactions with smooth muscle cells (9, 19). This review focuses on key ion channels expressed in UBSM cells and describes how they determine urinary bladder function. We highlight current understanding, recent advancements, and remaining gaps in knowledge.

ELECTRICAL ACTIVITY, INTRACELLULAR Ca²⁺, AND CONTRACTILITY OF UBSM

Membrane potential electrophysiological recordings from UBSM cells, either freshly isolated single cells (whole cell patch-clamp) or muscle fibers/tissue strips (sharp intracellular microelectrode), have revealed various types of electrical activity. Single UBSM cells, when measured with the perforated whole cell current-clamp technique (current = 0 mode), exhibited electrical quiescence, regular or irregular spontaneous transient hyperpolarizations (STHs) of variable amplitude, slow depolarizations, and action potentials (128, 130). The utility of the perforated patch-clamp technique ensured optimum preservation of intracellular cell composition, as the amphotericin (or nystatin)-formed pores (inserted into the plasma membrane at the tip of a patch electrode) primarily allow flux of monovalent cations but not Ca²⁺ or intracellular proteins. Similarly, when a sharp microelectrode technique has been used to record from a single UBSM cell in an isolated muscle bundle or tissue strip preparation, the activities measured encompassed electrical quiescence, slow depolarizations, bursts of either regular or irregular, fast action potentials, and high-frequency fast action potentials (9, 19, 52). Just as in the case of neurons, multiple voltage-sensitive ion channels, including Ca²⁺- and K⁺-selective channel subtypes, determine the UBSM electrical activity encompassing action potential generation. A notable exception is the absence of tetrodotoxin-sensitive voltage-gated Na⁺ (Na_v) channels (85). Pre-action potential slow depolarization and the fast upstroke phases depend on voltage-gated Ca²⁺ (Ca_v) channels (9, 19). The net Ca²⁺ influx via Ca_v channels leads to a global intracellular Ca²⁺ concentration increase. The elevated Ca²⁺ causes contraction through a series of biochemical events, including the involvement of Ca²⁺-calmodulin and myosin light-chain kinase (9, 19). On the other hand, inhibition of Ca_v channels by hyperpolarization mediated by either activation of K⁺ channels or inhibition of nonselective cation channels leads to a decrease of intracellular Ca²⁺ and subsequent UBSM relaxation (9, 19).

An important advancement in our understanding of urinary bladder physiology has been achieved by a series of highly sophisticated experiments in which simultaneous recordings of electrical activity, intracellular Ca²⁺ concentration, and contractility were made in UBSM preparations from the guinea pig, pig, and human (52–54, 163). These studies revealed the temporal sequence of fast action potential upstroke, rapidly rising Ca²⁺ transient with a rather long duration, and development of a relatively slow phasic contraction event. Although this fundamental contraction-generating mechanism has been found in all species studied, considerable interspecies differences have been noted in action potential shapes, profiles of Ca²⁺ transients, and related phasic contractions (19, 52–54, 163). While key contributing factors remain to be established, differential ion channel expressions or regulatory mechanisms likely play a role. This has led to the conclusion that although studies on animal models are very informative, they are not a substitute for conducting and determining responses of human UBSM tissues and single cells.

Experimentally, spontaneous phasic contractions have been recorded in mucosa-free isolated UBSM strips in vitro from human and animal (e.g., guinea pig and rat) preparations. They also have been visualized by in vivo cystometry or urodynamic assessments of intravesical pressure measured from rats or humans (9, 19). A notable observation from these methods was the discovery of spontaneous nonvoiding contractions; the significance of these continues to be debated in the scientific community (58). It has been proposed that detrusor overactivity (DO) due to excessive nonvoiding contractions in the bladder underlies at least some forms of overactive bladder (OAB) and lower urinary tract symptoms (LUTS) (9, 10, 19). Indeed, UBSM cells obtained from human bladders with OAB displayed aberrant Ca²⁺ oscillations and elevated intracellular Ca²⁺ concentrations, supporting intrinsic dysfunction (158).

In UBSM cells and tissue, membrane potential and various types of electrical activity, including the spontaneous intrinsic action potential, depend on multiple voltage-sensitive (or voltage-gated) and voltage-insensitive ion channels. Ca²⁺, K⁺, transient receptor potential (TRP), and Cl⁻ channel types have been identified as key determinants in UBSM (Fig. 1). The sections below review the roles of each channel type and highlight key supporting lines of evidence.

Fig. 1.

Key urinary bladder smooth muscle cell ion channels involved in the regulation of excitability and contractility. Shown are ion channels that 1) promote excitation via depolarization [Ca_v1.2 (L-type), Ca_v3 (T-type), TRPM4, TRPV4, TRPV2, and TRPC4/6], displayed in green; 2) reduce excitation via hyperpolarization [K_Ca1.1 (BK), K_v7 (KCNQ), K_v2, K_v1, K_ir6-SUR (K_ATP), K_Ca2 (SK), and K_2P], displayed in red; and 3) could play either role depending on the urinary bladder smooth muscle (UBSM) cell state [Cl_v and TMEM16A/Ano1; the question mark (?) after TMEM16/Ano1 refers to an uncertain and controversial role of this ion channel type in UBSM]. Solid black arrows indicate net ion flux across the UBSM cell membrane; dashed black arrows are for Ca²⁺ release from the sarcoplasmic reticulum (SR) via either ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP₃Rs). Ca²⁺ influx via Ca_v1.2 channels, regulated by UBSM cell depolarization or hyperpolarization, increases cytosolic Ca²⁺ concentration that subsequently triggers UBSM contraction. IP₃R-mediated Ca²⁺ release from the SR also contributes to intracellular Ca²⁺ concentration. Please see text and Tables 1–5 for detailed descriptions of each type of ion channel in UBSM. Ca_v, voltage-gated Ca²⁺; TRPM, transient receptor potential (TRP) melastatin; TRPV, TRP vanilloid; TRPC, TRP canonical; K_Ca, Ca²⁺-activated K⁺; K_v, voltage-gated K⁺; K_ir, inward-rectifying K⁺; K_2P, two-pore domain K⁺; Cl_v, voltage-gated Cl⁻.

UBSM Ca_v CHANNELS

Ca_v channels are critically important for the excitation-contraction coupling in UBSM (Fig. 1). They underlie the depolarization phase of the action potential that leads to an increase in the global intracellular Ca²⁺ concentration and subsequent generation of UBSM phasic contractions (52, 57). Ca_v1.2 (L-type, α_1C), Ca_v3.1 (T-type, α_1G), and Ca_v3.3 (T-type, α_1I) have been detected at mRNA and/or protein levels in UBSM of various species (Fig. 1 and Table 1). Since expression in whole UBSM tissue can originate from all cell types (including interstitial cells, vascular, nerve fibers, etc.) present in the tissue preparation, studies in UBSM cells only (i.e., isolated myocytes) have definitely concluded the presence of a given Ca_v channel in UBSM cells. As of now, expression studies of Ca_v channels in isolated UBSM cells, including from humans, are lacking.

Table 1.

Expression of Ca_v channels in UBSM whole tissues and isolated cells

Type	Detection	Preparation	Finding/Detection	Reference(s)
Cav1.1 (CACNA1S, α1S)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+) and increased following pBOO	Igawa et al. (76)
Cav1.2 (CACNA1C, α1C)	Protein	Human UBSM tissue	IHC (+) in human UBSM cells	Tomoda et al. (171)
Cav1.2 (CACNA1C, α1C)	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (+) and no change in the presence of FBS (proliferative agent)	Jiang et al. (80)
Cav1.2 (CACNA1C, α1C)	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+) and no change in db/db mice (type II diabetes model); quantitative RT-PCR (+) and increased in the streptozotocin model of diabetes	Wegener et al. (174); Jiang et al. (80); Leiria et al. (98)
Cav1.2 (CACNA1C, α1C)	Protein	Mouse UBSM tissue	WB (+)	Wegener et al. (174)
Cav1.2 (CACNA1C, α1C)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+) and decreased following pBOO	Igawa et al. (76)
Cav1.3 (CACNA1D, α1D)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+) and unchanged following pBOO	Igawa et al. (76)
Cav1.4 (CACNA1F, α1F)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+) and increased following pBOO	Igawa et al. (76)
Cav2.2 (CACNA1B, α1B)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+) and increased following pBOO	Igawa et al. (76)
Cav3.1 (CACNA1G, α1G)	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (+) and increased in the presence of FBS (proliferative agent)	Jiang et al. (80)
Cav3.1 (CACNA1G, α1G)	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+) and increased in db/db mice (type II diabetes model)	Jiang et al. (80)
Cav3.1 (CACNA1G, α1G)	mRNA	Rat UBSM tissue	RT-PCR (−) in controls and (+) increased following pBOO (semiquantitative analysis); quantitative RT-PCR (+) and nonsignificant increase/unchanged following pBOO	Li et al. (99); Igawa et al. (76)
Cav3.2 (CACNA1H, α1H)	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+) and no change in db/db mice (model of type II diabetes)	Jiang et al. (80)
Cav3.2 (CACNA1H, α1H)	mRNA	Rat UBSM tissue	RT-PCR (−) and undetected following pBOO; quantitative RT-PCR (+) and increased following pBOO	Li et al. (99); Igawa et al. (76)
Cav3.3 (CACNA1I, α1I)	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+) and no change in db/db mice (model of type II diabetes)	Jiang et al. (80)
Cav3.3 (CACNA1I, α1I)	mRNA	Rat UBSM tissue	RT-PCR (+) and increased following pBOO (semiquantitative analysis); quantitative RT-PCR (+) and increased following pBOO	Li et al. (99); Igawa et al. (76)

Ca_v, voltage-gated Ca²⁺; UBSM, urinary bladder smooth muscle; pBOO, partial bladder outflow obstruction; IHC, immunohistochemistry; WB, Western blot; (+), positive detection; (−), negative detection.

In contrast, functional electrophysiological observations on Ca_v channels and their properties in single UBSM cells have been reported. Original recordings of Ca_v1.2 currents in guinea pig UBSM cells were made as early as 1985, using what was at the time the newly developed patch-clamp technique (85, 86), and subsequently further characterized (108, 110, 111, 185). Similar to Ca_v1.2 channels in non-UBSM cells, UBSM Ca_v1.2 channels displayed maximum current activation at approximately −10 mV, multiple open channel states, pharmacological activation with the agonist Bay K8644, robust inhibition by dihydropyridines (e.g., nifedipine), and temperature sensitivity (108, 110, 111, 185). Blockade of Ca_V1.2 channels with dihydropyridines or other channel-selective inhibitors was shown to abolish spontaneous action potentials, associated Ca²⁺ transients, and related phasic contractions in UBSM (35, 52, 53, 57). Inducible and conditional smooth muscle-specific knockdown of Ca_v1.2 channels in mice resulted in severely reduced micturition, increased bladder mass, absence of spontaneous UBSM phasic contractions, and reduced high K⁺- and carbachol-induced contractions (174).

An important consideration for Ca_v1.2 channels is their regulation by G protein-coupled receptors and subsequently recruited intracellular signaling pathways. In UBSM, muscarinic (M) receptor stimulation with carbachol decreased Ca_v1.2 currents (188), while β-adrenoceptor (β-AR) activation (isoproterenol) could induce a slight enhancement in Ca_v1.2 channel activity (134, 153). The effects of isoproterenol on Ca_v1.2 currents remain controversial, as other studies found either no effect or a decrease (88, 89). The discrepancies likely reflect differences in experimental recording conditions used by distinct studies, possibly involving the specific recording method used (conventional vs. perforated whole cell patch-clamp, Ba²⁺ vs. Ca²⁺ in the extracellular solution for influx) and voltage-step protocols (favoring activation vs. inactivation).

Ca_v1.2 channel dysfunction/dysregulation may contribute to the development of LUTS based on findings in animal models. In UBSM of streptozotocin (STZ)-induced diabetic mice (a model of type I diabetes), mRNA expression of Ca_v1.2 (α_1C) was increased compared with controls (without STZ induction) (98). This channel expression increase in STZ-induced diabetic mice was associated with abnormal urinary bladder function, including increases in nonvoiding contractions (frequency and peak amplitude), micturition frequency, and peak pressure (all determined with cystometry) and enhanced UBSM contractility in vitro (98). Rats exhibiting DO due to partial bladder outflow obstruction (pBOO) showed higher mRNA expression of Ca_v1.1 and Ca_v1.4 in whole urinary bladder and UBSM tissue (76). In the same study (76), Ca_v1.2 mRNA levels showed only a slight decrease under pBOO. Since determinations were not made on isolated UBSM cells, the cellular origin of the detected expression for Ca_v1 channels remains uncertain (76).

Ca_v3 (T-type) channels, which normally activate at more negative potentials than Ca_v1.2 channels, have also been found to be expressed in UBSM, but their functional role remains unclear (Fig. 1) (42). Most likely, they contribute to the slight slow depolarizations known as “prepotentials” that precede the action potential spike. Ca_v3 channel opening thus appears to promote cell excitability and to favor the activation of Ca_v1.2 channels (Fig. 1). The potential functional roles of Ca_v3 channels in the lower urinary tract, including UBSM, are the subject of a past review [please see Fry and Jabr (42)]. Of note, Ca_v3 channels may play a role in LUTS. Experimentally induced DO in rats (pBOO model) was found to be linked to enhanced mRNA expression of Ca_v3.2 and Ca_v3.3 channels in UBSM and increased Ca_v3 (T-type) channel currents (76, 99). The novel pan-selective Ca_v3 channel inhibitor RQ-00311610 enhanced bladder capacity and voided volume in pBOO rats; hence, it displayed a favorable in vivo profile consistent with the blocker-induced reduction of UBSM excitability and contractility (99). UBSM cells obtained from patient donors exhibiting DO also showed increased Ca_v3 currents (159). Thus, Ca_v channels are critical regulators of UBSM function and may be pharmacologically targeted for LUTS indications.

In light of the importance of Ca_v channels in UBSM, it is surprising how little research activity has been undertaken, especially recently, to advance the following gaps in knowledge: 1) the understanding of their roles, specifically in human UBSM (importantly, single myocytes); 2) epigenetic regulatory mechanisms involving transcription and translation regulatory pathways; 3) intracellular channel trafficking; 4) UBSM cell type diversity; 5) regulation of nonexcitatory cellular functions (e.g., proliferation); 6) genetic manipulations of Ca_v channel subunits and their regulatory molecules and stimuli (for example, CRISPR-Cas9 and optogenetics); 7) interactions, either directly or indirectly, with other ion channels, receptors, and intracellular signaling molecules (including microRNA and lncRNA); and 8) the expression and functional consequences of such interacting units in health and urinary bladder diseases. The majority of the above concepts and current gaps in knowledge also apply to other ion channels as described further below.

UBSM K⁺ CHANNELS

The central role played by K⁺ channels in modulating UBSM function derives from their functionally antagonistic relationship with Ca_v channels (Fig. 1). Overall, K⁺ channels fine-tune UBSM excitability by controlling Ca_v channel activity, Ca²⁺ entry into UBSM cells, and therefore UBSM contractility. In general, activation of K⁺ channels leads to hyperpolarization and subsequent decreases of excitability and contractility via attenuation of Ca_v channel activity. In contrast, inhibition of K⁺ channels causes depolarization, promotes Ca_v channel opening, and enhances excitability and contractility. Two recent reviews have provided detailed descriptions of the topic of K⁺ channels in UBSM (128, 130).

There are four major families of K⁺ channels in UBSM: K_v channels, Ca²⁺-activated K⁺ (K_Ca) channels, inward-rectifying ATP-sensitive K⁺ (K_ir, K_ATP) channels, and the two-pore domain K⁺ (K_2P) channel family (Fig. 1).

The K_v channel family in the human genome is represented by 40 genes classified into 12 subfamilies based on sequence homology (129, 130). By convention, K_v channels use a specific nomenclature where the number after “K_v” indicates the gene subfamily (K_v1−K_v12) encoding a specific α-subunit (129, 130). The number after the dot specifies the individual K_v channel member (K_v1.1, K_v1.2, K_v1.3, etc.), and splice variant isoforms are indicated by lowercase letters at the end (K_v1.1a, K_v1.1b, etc.). K_Ca (K_Ca1-K_Ca5), K_ir (K_ir1−K_ir7), and K_2P (K_2P1−K_2P15) channels use the same classification (129, 130).

Structurally, an assembly of four α-subunits of the same type forms a functional K⁺ channel (129, 130). The K⁺ channel pore-forming α-subunit can have 6S transmembrane (K_v channels), 6S or 7S transmembrane (K_Ca), 2S transmembrane (K_ir and K_ATP channels), or 4S transmembrane (K_2P) architecture (129, 130). K_v1−K_v4, K_v7, and K_v10−K_v12 channels form homotetrameric channels. K_v5, K_v6, K_v8, and K_v9 channels do not assemble as homotetramers but rather form heterotetrameric K_v channels with K_v2 subunits (129, 130). In addition, accessory regulatory subunits such as β or KCNE subunits can associate with α-subunits to fine-tune the electrophysiological and pharmacological properties of K⁺ channel complexes (129, 130).

UBSM K_v Channels

The functional role of K_v channels in UBSM is a hot topic subject to increasing investigation (Fig. 1) (30, 55, 66, 68, 140). In UBSM cells, K_v channels work to repolarize the membrane potential to end the action potential and also have a role in setting the resting membrane potential (52, 55, 66, 68, 130, 169). Expression of multiple K_v channel subtypes in UBSM tissues (without mucosa) and cells (myocytes) at mRNA and protein levels have been reported in the literature (Table 2). Although certain K_v channel members (e.g., K_v2.1 and K_v7.4) were expressed uniformly in UBSM of different species, others showed differential detection profiles. For example, rat and mouse UBSM cells expressed K_v2.2 channels, but guinea pig and human UBSM cells did not (Table 2). In mouse UBSM, K_v2.1 subunit expression was associated with electrically silent subunits (K_v5.1, K_v6.1, K_v6.2, or K_v6.3) likely forming heterotetramers with K_v2.1 channels (K_v2.1/X channels) (169). Similarly to rat UBSM (30), human UBSM cells had K_v2.1 and K_v2.2 channels along with the electrically silent K_v9.1 subunit (66). Guinea pig UBSM expressed K_v2.1 (but not K_v2.2) and electrically silent K_v6.2, K_v6.3, K_v8.2, and K_v9.1–K_v9.3 subunits, indicating marked species differences (68). Human, rat, and guinea pig UBSM tissues did not contain the K_v4.2 channel, which mediates fast A-type K_v current (30, 66, 68). A functional role for the K_v11.1 channel has also been proposed in guinea pig UBSM (77).

Table 2.

Expression of K_v channels and KCNE subunits in UBSM whole tissues and isolated cells

Type	Detection	Preparation	Method/Finding	Reference(s)
Kv1.1 (KCNA1)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (120)
Kv1.2 (KCNA2)	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (120)
Kv1.3 (KCNA3)	Protein	Human UBSM tissue	IHC (+) in UBSM cells; WB (+)	Davies et al. (36)
Kv1.5 (KCNA5)	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (120)
Kv1.6 (KCNA6)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (120)
Kv1.6 (KCNA6)	Protein	Human UBSM tissue	IHC (+) in UBSM cells; WB (+)	Davies et al. (36)
Kv2.1 (KCNB1)	mRNA	Human UBSM tissue	RT-PCR (+)	Hristov et al. (66)
Kv2.1 (KCNB1)	Protein	Human UBSM tissue	WB (+)	Hristov et al. (66)
Kv2.1 (KCNB1)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (66)
Kv2.1 (KCNB1)	Protein	Human UBSM cells	ICC (+)	Hristov et al. (66)
Kv2.1 (KCNB1)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169); Layne et al. (94)
Kv2.1 (KCNB1)	mRNA	Mouse UBSM cells	RT-PCR (+)	Thorneloe and Nelson (169)
Kv2.1 (KCNB1)	mRNA	Rat UBSM tissue	RT-PCR (+)	Chen et al. (30); Ohya et al. (120)
Kv2.1 (KCNB1)	Protein	Rat UBSM tissue	WB (+)	Chen et al. (30)
Kv2.1 (KCNB1)	mRNA	Rat UBSM cells	RT-PCR (+); RT-PCR (+) decrease in expression following spinal cord injury (semiquantitative analysis)	Chen et al. (30); Gan et al. (44)
Kv2.1 (KCNB1)	Protein	Rat UBSM cells	ICC (+)	Chen et al. (30)
Kv2.1 (KCNB1)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv2.1 (KCNB1)	Protein	Guinea pig UBSM tissue	WB (+)	Hristov et al. (68)
Kv2.1 (KCNB1)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv2.1 (KCNB1)	Protein	Guinea pig UBSM cells	ICC (+)	Hristov et al. (68)
Kv2.2 (KCNB2)	mRNA	Human UBSM tissue	RT-PCR (+)	Hristov et al. (66)
Kv2.2 (KCNB2)	Protein	Human UBSM tissue	WB (+)	Hristov et al. (66)
Kv2.2 (KCNB2)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (66)
Kv2.2 (KCNB2)	Protein	Human UBSM cells	ICC (+)	Hristov et al. (66)
Kv2.2 (KCNB2)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169)
Kv2.2 (KCNB2)	mRNA	Mouse UBSM cells	RT-PCR (−)	Thorneloe and Nelson (169)
Kv2.2 (KCNB2)	mRNA	Rat UBSM tissue	RT-PCR (+); RT-PCR (−)	Chen et al. (30); Ohya et al. (120)
Kv2.2 (KCNB2)	Protein	Rat UBSM tissue	WB (+)	Chen et al. (30)
Kv2.2 (KCNB2)	mRNA	Rat UBSM cells	RT-PCR (+); RT-PCR (−)	Chen et al. (30); Gan et al. (44)
Kv2.2 (KCNB2)	Protein	Rat UBSM cells	ICC (+)	Chen et al. (30)
Kv2.2 (KCNB2)	mRNA	Guinea pig UBSM tissue	RT-PCR (−)	Hristov et al. (68)
Kv2.2 (KCNB2)	Protein	Guinea pig UBSM tissue	WB (−)	Hristov et al. (68)
Kv2.2 (KCNB2)	mRNA	Guinea pig UBSM cells	RT-PCR (−)	Hristov et al. (68)
Kv2.2 (KCNB2)	Protein	Guinea pig UBSM cell	ICC (−)	Hristov et al. (68)
Kv3.1 (KCNC1)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (120)
Kv3.2 (KCNC2)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (120)
Kv4.2 (KCND2)	mRNA	Human UBSM tissue	RT-PCR (+)	Hristov et al. (66)
Kv4.2 (KCND2)	Protein	Human UBSM tissue	WB (−)	Hristov et al. (66)
Kv4.2 (KCND2)	mRNA	Human UBSM cells	RT-PCR (−)	Hristov et al. (66)
Kv4.2 (KCND2)	Protein	Human pig UBSM tissue	WB (−)	Hristov et al. (66)
Kv4.2 (KCND2)	mRNA	Guinea pig UBSM tissue	RT-PCR (−)	Hristov et al. (68)
Kv4.2 (KCND2)	Protein	Guinea pig UBSM tissue	WB (−)	Hristov et al. (68)
Kv4.2 (KCND2)	mRNA	Guinea pig UBSM cells	RT-PCR (−)	Hristov et al. (68)
Kv4.2 (KCND2)	Protein	Guinea pig UBSM cells	ICC (−)	Hristov et al. (68)
Kv4.2 (KCND2)	mRNA	Rat UBSM tissue	RT-PCR (−)	Chen et al. (30)
Kv4.2 (KCND2)	Protein	Rat UBSM tissue	WB (−)	Chen et al. (30)
Kv4.2 (KCND2)	mRNA	Rat UBSM cells	RT-PCR (−)	Chen et al. (30)
Kv4.2 (KCND2)	Protein	Rat UBSM cells	ICC (−)	Chen et al. (30)
Kv4.3 (KCND3)	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (119)
Kv5.1 (KCNF1)	mRNA	Guinea pig UBSM tissue	RT-PCR (−)	Hristov et al. (68)
Kv5.1 (KCNF1)	mRNA	Guinea pig UBSM cells	RT-PCR (−)	Hristov et al. (68)
Kv5.1 (KCNF1)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169)
Kv5.1 (KCNF1)	mRNA	Mouse UBSM cells	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.1 (KCNG1)	mRNA	Guinea pig UBSM tissue	RT-PCR (−)	Hristov et al. (68)
Kv6.1 (KCNG1)	mRNA	Guinea pig UBSM cells	RT-PCR (−)	Hristov et al. (68)
Kv6.1 (KCNG1)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.1 (KCNG1)	mRNA	Mouse UBSM cells	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.2 (KCNG2)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv6.2 (KCNG2)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv6.2 (KCNG2)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.2 (KCNG2)	mRNA	Mouse UBSM cells	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.3 (KCNG3)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv6.3 (KCNG3)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv6.3 (KCNG3)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Thorneloe and Nelson (169)
Kv6.3 (KCNG3)	mRNA	Mouse UBSM cells	RT-PCR (+)	Thorneloe and Nelson (169)
Kv7.1 (KCNQ1)	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), weak relative expression; quantitative RT-PCR (+) and moderate relative expression, level increased in patients with pBOO	Bientinesi et al. (16); Svalo et al. (162)
Kv7.1 (KCNQ1)	Protein	Human UBSM tissue	WB (+)	Bientinesi et al. (16)
Kv7.1 (KCNQ1)	mRNA	Guinea pig UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Afeli et al. (2)
Kv7.1 (KCNQ1)	Protein	Guinea pig UBSM tissue	IHC (+) in UBSM cells	Afeli et al. (2); Anderson et al. (8)
Kv7.1 (KCNQ1)	mRNA	Guinea pig UBSM cells	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Afeli et al. (2); Anderson et al. (8)
Kv7.1 (KCNQ1)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+), weak relative expression; RT-PCR (−)	Svalo et al. (161); Ohya et al. (117)
Kv7.2 (KCNQ2)	mRNA	Human UBSM tissue	Quantitative RT-PCR (+/−), weak/negligible relative expression; quantitative RT-PCR (+/−), weak/negligible relative expression, not detected in patients with pBOO; quantitative RT-PCR (+) with weak relative expression	Bientinesi et al. (16); Svalo et al. (162) Seefeld et al. (146)
Kv7.2 (KCNQ2)	mRNA	Guinea pig UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Afeli et al. (2)
Kv7.2 (KCNQ2)	Protein	Guinea pig UBSM tissue	IHC (+) in UBSM cells; WB (+)	Afeli et al. (2); Anderson et al. (8); Provence et al. (140)
Kv7.2 (KCNQ2)	mRNA	Guinea pig UBSM cells	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Afeli et al. (2); Anderson et al. (8)
Kv7.2 (KCNQ2)	Protein	Guinea pig UBSM cells	ICC (+)	Provence et al. (140)
Kv7.2 (KCNQ2)	mRNA	Rat UBSM tissue	RT-PCR (−); quantitative RT-PCR (−)	Ohya et al. (117); Svalo et al. (161)
Kv7.2 (KCNQ2)	Protein	Rat UBSM tissue	WB (−)	Svalo et al. (161)
Kv7.3 (KCNQ3)	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), weak relative expression; quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO; quantitative RT-PCR (+), moderate relative expression	Bientinesi et al. (16); Svalo et al. (162); Seefeld et al. (146)
Kv7.3 (KCNQ3)	mRNA	Guinea pig UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), moderate relative expression	Afeli et al. (2)
Kv7.3 (KCNQ3)	Protein	Guinea pig UBSM tissue	IHC (+) in UBSM cells; WB (+)	Afeli et al. (2); Anderson et al. (8); Provence et al. (140)
Kv7.3 (KCNQ3)	mRNA	Guinea pig UBSM cells	RT-PCR (+); quantitative RT-PCR (+), low relative expression	Afeli et al. (2); Anderson et al. (8)
Kv7.3 (KCNQ3)	Protein	Guinea pig UBSM cells	ICC (+)	Provence et al. (140)
Kv7.3 (KCNQ3)	mRNA	Rat UBSM tissue	RT-PCR (−); quantitative RT-PCR (−)	Ohya et al. (117); Svalo et al. (161)
Kv7.3 (KCNQ3)	mRNA	Pig UBSM tissue	Quantitative RT-PCR (+/−), very weak relative expression	Svalo et al. (160)
Kv7.4 (KCNQ4)	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), high relative expression; quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO	Bientinesi et al. (16); Svalo et al. (162); Seefeld et al. (146)
Kv7.4 (KCNQ4)	Protein	Human UBSM tissue	WB (+)	Bientinesi et al. (16)
Kv7.4 (KCNQ4)	mRNA	Guinea pig UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low relative expression	Afeli et al. (2)
Kv7.4 (KCNQ4)	Protein	Guinea pig UBSM tissue	IHC (−) in UBSM cells; IHC (+) in UBSM cells	Afeli et al. (2); Anderson et al. (8)
Kv7.4 (KCNQ4)	mRNA	Guinea pig UBSM cells	RT-PCR (−); RT-PCR (+); quantitative RT-PCR (+), low relative expression	Afeli et al. (2); Anderson et al. (7)
Kv7.4 (KCNQ4)	Protein	Guinea pig UBSM cells	In situ PLA (+), detected as heteromers with Kv7.5	Provence et al. (138)
Kv7.4 (KCNQ4)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+), most highly expressed Kv7 subtype	Svalo et al. (161)
Kv7.4 (KCNQ4)	Protein	Rat UBSM tissue	WB (+)	Svalo et al. (161)
Kv7.4 (KCNQ4)	mRNA	Pig UBSM tissue	Quantitative RT-PCR (+), high relative expression	Svalo et al. (160)
Kv7.5 (KCNQ5)	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), weak relative expression; quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO; quantitative RT-PCR (+), high relative expression	Bientinesi et al. (16); Svalo et al. (162); Seefeld et al. (146)
Kv7.5 (KCNQ5)	Protein	Human UBSM tissue	WB (+)	Bientinesi et al. (16)
Kv7.5 (KCNQ5)	mRNA	Guinea pig UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), moderate relative expression	Afeli et al. (2)
Kv7.5 (KCNQ5)	Protein	Guinea pig UBSM tissue	IHC (+) in UBSM cells	Afeli et al. (2); Anderson et al. (8)
Kv7.5 (KCNQ5)	mRNA	Guinea pig UBSM cells	RT-PCR (+); quantitative RT-PCR (+), moderate relative expression	Afeli et al. (2); Anderson et al. (8)
Kv7.5 (KCNQ4)	Protein	Guinea pig UBSM cells	In situ PLA (+), detected as heteromers with Kv7.4	Provence et al. (138)
Kv7.5 (KCNQ5)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+), low relative expression	Svalo et al. (161)
Kv7.5 (KCNQ5)	mRNA	Pig UBSM tissue	Quantitative RT-PCR (+), low relative expression	Svalo et al. (160)
Kv8.1 (KCNV1)	mRNA	Guinea pig UBSM tissue	RT-PCR (−)	Hristov et al. (68)
Kv8.1 (KCNV1)	mRNA	Guinea pig UBSM cells	RT-PCR (−)	Hristov et al. (68)
Kv8.2 (KCNV2)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv8.2 (KCNV2)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv9.1 (KCNS1)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv9.1 (KCNS1)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv9.2 (KCNS2)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv9.2 (KCNS2)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv9.3 (KCNS3)	mRNA	Human UBSM tissue	RT-PCR (+)	Hristov et al. (66)
Kv9.3 (KCNS3)	Protein	Human UBSM tissue	WB (−)	Hristov et al. (66)
Kv9.3 (KCNS3)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (66)
Kv9.3 (KCNS3)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Hristov et al. (68)
Kv9.3 (KCNS3)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Hristov et al. (68)
Kv9.3 (KCNS3)	mRNA	Rat UBSM tissue	RT-PCR (+)	Chen et al. (30); Ohya et al. (120)
Kv9.3 (KCNS3)	Protein	Rat UBSM tissue	WB (−)	Chen et al. (30)
Kv9.3 (KCNS3)	mRNA	Rat UBSM cells	RT-PCR (+)	Chen et al. (30)
Kv11.1 (KCNH2, ERG1)	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (117)
Kv11.1 (KCNH2, ERG1)	Protein	Rat UBSM tissue	WB (+)	Ohya et al. (117)
Kv11.2 (KCNH6, ERG2)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (117)
Kv11.3 (KCNH7, ERG3)	mRNA	Rat UBSM tissue	RT-PCR (−)	Ohya et al. (117)
KCNE1	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), moderate relative expression, level unchanged/nonsignificant decrease in patients with pBOO	Svalo et al. (162)
KCNE1	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (117)
KCNE1	Protein	Rat UBSM tissue	WB (+)	Ohya et al. (117)
KCNE2	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO	Svalo et al. (162)
KCNE2	mRNA	Rat UBSM tissue	RT-PCR (+)	Ohya et al. (117)
KCNE3	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO	Svalo et al. (162)
KCNE4	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO	Svalo et al. (162)
KCNE5	mRNA	Human UBSM tissue	Quantitative RT-PCR (+), moderate relative expression, level unchanged in patients with pBOO	Svalo et al. (162)

K_v, voltage-gated K⁺; UBSM, urinary bladder smooth muscle; IHC, immunohistochemistry; WB, Western blot; ICC, immunocytochemistry; pBOO, partial bladder outflow obstruction; PLA, in situ proximity ligation assay; (+), positive detection; (−), negative detection; (+/−), very low/borderline detection.

More recently, K_v7 channel members have been found in UBSM cells and tissues (2, 8, 143, 161). KCNQ1–KCNQ5 genes encode K_v7.1–K_v7.5 channels, specifically their α-subunits (129, 130). K_v7.4 channels have been detected in UBSM cells and/or tissues of guinea pigs, pigs, and rats (Table 2). Although an initial report (2) found an absence of K_v7.4 in guinea pig UBSM, other studies (8, 138) showed positive detections and, thus, confirmed its presence. K_v7 channel pore-forming α-subunits can further associate with small, single transmembrane regulatory subunits encoded by KCNE genes, which are expressed in UBSM (Table 2). Hence, the diversity of K_v7 channel heteromultimeric combinations, coupled with accessory KCNE subunits, expands the possible combinations expressed in UBSM. In guinea pig UBSM cells, native K_v7 channels comprise K_v7.2/K_v7.3 and K_v7.4/K_v7.5 α-subunit combinations, but K_v7.3/K_v7.5 channel heteromers and homomeric combinations could also be present (138, 140). The absence of K_v7.2 channels in rat UBSM and their relatively very low or negligible expression detected in human UBSM imply that the classical neuron-specific K_v7.2/K_v7.3 heteromers do not have a functional role in urinary bladders of these species (Table 2). Studies on human UBSM tissues are, however, very limited, and those on UBSM cells are still missing in the literature.

One of the advantages of studying the K_v channel family is the availability of selective or preferential channel subtype modulators along with broad nonselective tools (129, 130). They have allowed the initiation of studies elucidating the role of K_v channels in UBSM. One such K_v channel modulator, stromatoxin-1 (which is preferentially selective for K_v2.1, K_v2.2, K_v4.2, and K_v2.1/K_v9.3 channels) attenuated voltage-step induced currents (voltage-clamp) and induced depolarization (current-clamp) in human and guinea pig UBSM cells (voltage-clamp) and increased UBSM contractility in vitro (66, 68). Stromatoxin-1 also enhanced contractility of rat UBSM (30).

K_v7 channel pharmacological modulators include the activators retigabine (selective for K_v7.2–K_v7.5 channels) and L-364,373 (selective for K_v7.1 channels) as well as the K_v7 channel inhibitors XE991 and linopiridine (both blocking K_v7.1–K_v7.5 channels) (8, 140). Additional novel K_v7 channel subtype-selective openers, such as ICA-069673 (K_v7.2/K_v7.3) and ML213 (K_v7.2 and K_v7.4), have been recently shown to reduce UBSM excitability and contractility (140, 160, 162). Retigabine (Ezogabine or Trobalt), the first-in-class K_v7 channel opener developed for epilepsy, increased micturition volume and voiding intervals; when given intravesically, this compound decreased capsaicin-induced DO (157). In isolated UBSM strips, retigabine reduced spontaneous, pharmacologically induced, and nerve-evoked UBSM contractions, and the effects were reversed by the K_v7 channel inhibitor XE991 (16, 143, 160, 162). The reported increased incidence of urinary retention in retigabine-treated human subjects compared with placebo treatment in clinical trials for epilepsy (20) further supported the concept that targeting K_v7 channels via a selective opener might be a very useful new pharmacological approach for the treatment of OAB. Thus, what was initially identified as a clinical side effect for retigabine may actually lead to novel therapeutics for OAB.

Experimental findings suggest dysregulation of K_v channels in urinary bladder dysfunction, although data are very limited. Rats with UBSM hyperreflexia induced by spinal cord injury showed decreased expression of mRNA for the K_v2.1 channel in UBSM, but the study lacked quantitative evaluation (44). UBSM obtained from patient donors with pBOO due to benign prostatic hyperplasia (BPH) showed increased mRNA expression of the K_v7.1 channel, most likely reflecting a compensatory mechanism for reducing overexcitability due to DO (162). Consistent with the presence of K_v7 channel subtypes in pBOO-BPH UBSM tissues, the K_v7 channel activators retigabine and ML213 induced inhibitions of an M receptor agonist and 20 mM K⁺-induced contractions (162). This finding supports that small-molecule K_v7 channel openers can potentially be used as a therapeutic approach for the management of OAB. Future investigations on K_v channels, including the role of K_v7 channel subtypes in various animal models of urinary bladder disease and in human UBSM cells/tissues from patient donors with and without OAB/DO, are critically needed.

A recent study (173) demonstrated that K_v7 channels regulate urinary bladder voiding via afferent neuronal signaling modulation, but a role for UBSM K_v7 channels could not be confirmed in mice. Whether K_v7 channels expressed in mouse UBSM cells contribute to urinary bladder regulation still remains to be resolved. At present, no molecular biology (mRNA or protein) expression report has yet described the presence or absence of any K_v7 channel subtypes directly in single UBSM cells. Functional contractility studies examining the effects of K_v7 channel modulation under physiological conditions (~5 mM extracellular K⁺ rather than elevated 60–80 mM) are also lacking. Although the K_v7 channel activator retigabine can directly inhibit Ca_v1.2 (L-type Ca²⁺) channels, the magnitude of inhibition is rather low compared with classical blockers such as dihydropyridine nifedipine or nicardipine (101, 173, 174). Furthermore, newer and more selective K_v7 channel modulators, ICA-069673 and ML213, given their lack of interaction with Ca_v1.2 channels, exhibit a more favorable pharmacological profile for the study of K_v7 channels (7, 189) and are yet to be examined in mouse UBSM function. In guinea pig, rat, and pig experiments, retigabine, ICA-069673, and ML213 induced UBSM relaxation that was dependent on the K⁺ driving force and displayed much weaker or no efficacy under 60 mM extracellular K⁺ compared with robust effects observed under 5 or 20 mM K⁺ (11, 138, 140, 143, 161). This key observation provided evidence that the underlying mechanism for retigabine, ML213, and ICA-069673 involved the opening of K_v7 channels, and this finding is further supported by attenuation of the effects of K_v7 channel activators by XE991 (138, 140, 143, 161). In mouse UBSM, the effects of retigabine and XE991 were studied only under high extracellular K⁺ and not on spontaneous phasic contractions under physiological extracellular K⁺ (173). Electrophysiological patch-clamp studies in single mouse UBSM cells conducted under the optimum experimental conditions required for recording of smooth muscle K_v7 channel currents are currently not available in the published literature. The well-established and appropriate protocol, described in detail elsewhere, involves the perforated patch-clamp approach, pharmacological isolation, use of a depolarized holding potential, and relatively long voltage steps (22, 138). The only patch-clamp study on mouse UBSM cells currently available did not use these optimized conditions (173). Unless proper experimental conditions are applied, the presence of other contributing K⁺ currents in UBSM cells complicates data interpretation, especially when examining the pharmacological effects of K_v7 channel modulators (173). Thus, future studies are required to clarify whether mouse UBSM cells express functional K_v7 channels and which specific K_v7 channel subtypes are involved in mouse UBSM regulation. Resolution of this dilemma will have an impact on the potential utility and relevance of K_v7 channel genetic mouse models, whether global or smooth muscle specific, for the study of UBSM function. As initially suggested by Tykocki et al. (173), mouse UBSM cells may not express functional K_v7 channels, unlike other species including the guinea pig, rat, or human, where UBSM K_v7 channel expression and function have already been established (2, 11, 133, 138, 140). Indeed, retigabine failed to hyperpolarize the membrane potential of single UBSM cells in mice, but this activator did so effectively in guinea pigs, rats, and humans (2, 11, 173). The efficacy of retigabine to induce hyperpolarization in UBSM cells may also depend on the resting membrane potential. Indeed, under the experimental recording conditions favoring a more hyperpolarized state (approximately −40 mV, mouse UBSM cells), retigabine lacked efficacy (173). In contrast, at approximately −20 mV, retigabine as well as other K_v7 channel activators did induce hyperpolarization in guinea pig UBSM cells (2, 138, 140). Collectively, the area of UBSM K_v7 channel expression and function is currently one of the most interesting and evolving topics in the field of translational urological research.

UBSM K_Ca Channels: K_Ca1.1, K_Ca2.x, and K_Ca3.1

In UBSM, the K_Ca channel family (Fig. 1) is represented by three major subtypes of channels, which are classified based on their single channel conductance: large-conductance (100–300 pS) voltage- and Ca²⁺-activated K⁺ (K_Ca1.1 or BK) channels, intermediate-conductance (20–80 pS) Ca²⁺-activated K⁺ (K_Ca3.1 or IK) channels, and small-conductance (~10 pS) Ca²⁺-activated K⁺ channels (K_Ca2.1−K_Ca2.3/SK1–SK3) (130). K_Ca2 and K_Ca3.1 channels are Ca²⁺ activated but voltage insensitive (129, 130).

UBSM K_Ca1.1 channels.

Among all K⁺ channels, the K_Ca1.1 channel (Fig. 1) is recognized as the most prominent and physiologically relevant regulator of UBSM function in health and disease (21, 55, 57, 60–62, 67, 106, 131, 134, 155, 156, 168). Besides its high single channel conductance (~100–200 pS), the property of dual activations by depolarization and intracellular Ca²⁺, unique among K⁺ channels, positions the K_Ca1.1 channel as a Ca²⁺/voltage signal integrator in the modulation of UBSM cell excitability (67, 104, 129, 130). Physiologically, K_Ca1.1 channels provide a negative feedback mechanism to limit the amplitude and duration of UBSM action potentials and related phasic contractions (53, 57, 60, 67). The K_Ca1.1 channel has very high expression levels in UBSM (31, 67, 116, 131) in all species studied (Table 3). In recent years, methodological identification and characterization of K_Ca1.1 channels have advanced knowledge of their functional and regulatory roles in native human UBSM and have also identified a potential role in urinary bladder diseases (4, 65, 67, 71, 104, 178, 181, 183).

Table 3.

Expression of K_Ca channels in UBSM whole tissues and isolated cells

Type	Detection	Preparation	Method/Finding	Reference(s)
KCa1.1 α1 (KCNMA1)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), decrease in patients with NDO; quantitative RT-PCR (+), decrease in patients with BPH-DO	Hristov et al. (67); Hristov et al. (65); Chang et al. (27)
KCa1.1 α1 (KCNMA1)	Protein	Human UBSM tissue	WB (+); WB (+), decrease in patients with BPH-DO	Hristov et al. (67); Chang et al. (27)
KCa1.1 α1 (KCNMA1)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (67)
KCa1.1 α1 (KCNMA1)	Protein	Human UBSM cells	ICC (+)	Hristov et al. (67)
KCa1.1 α1 (KCNMA1)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), unchanged expression in pBOO; quantitative RT-PCR (+), decrease in pBOO; RT-PCR (+) decrease in pBOO (semiquantitative)	Chen and Petkov (31); Kita et al. (84); Aydin et al. (12); Li et al. (100)
KCa1.1 α1 (KCNMA1)	mRNA	Rat UBSM tissue with mucosa	RT-PCR (+); quantitative RT-PCR (+), unchanged expression in pBOO	Kim and Yang (83); Kita et al. (84)
KCa1.1 α1 (KCNMA1)	Protein	Rat UBSM tissue with mucosa	WB (+)	Kim and Yang (83)
KCa1.1 α1 (KCNMA1)	mRNA	Rat UBSM cells	RT-PCR (+)	Chen and Petkov (31),
KCa1.1 α1 (KCNMA1)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Chen and Petkov (31); Layne et al. (94)
KCa1.1 α1 (KCNMA1)	mRNA	Mouse UBSM tissue with mucosa	RT-PCR (+)	Ohya et al. (118)
KCa1.1 α1 (KCNMA1)	Protein	Mouse UBSM tissue	WB (+); IHC (+) in UBSM cells	Meredith et al. (106); White et al. (176); Sprossmann et al. (156); Werner et al. (175)
KCa1.1 α1 (KCNMA1)	mRNA	Mouse UBSM cells	RT-PCR (+); quantitative RT-PCR (+), moderate relative expression	Chen and Petkov (31); Lee et al. (97)
KCa1.1 α1 (KCNMA1)	Protein	Rabbit UBSM tissue	WB (+), decrease in pBOO (semiquantitative); IHC (+) in UBSM cells, decrease in pBOO	Chang et al. (27)
KCa1.1 β1 (KCNMB1)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+) decrease in patients with BPH-DO; quantitative RT-PCR (+), unchanged in patients with NDO	Hristov et al. (67); Chang et al. (27); Hristov et al. (65)
KCa1.1 β1 (KCNMB1)	Protein	Human UBSM tissue	WB (+); WB (+), decrease in BPH-DO patients	Hristov et al. (67); Chang et al. (27)
KCa1.1 β1 (KCNMB1)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (67)
KCa1.1 β1 (KCNMB1)	Protein	Human UBSM cells	ICC (+)	Hristov et al. (67)
KCa1.1 β1 (KCNMB1)	mRNA	Cultured human UBSM cells	RT-PCR (+)	Chang et al. (27)
KCa1.1 β1 (KCNMB1)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), increase in pBOO	Chen and Petkov (31); Aydin et al. (12); Kita et al. (84)
KCa1.1 β1 (KCNMB1)	Protein	Rat UBSM tissue	WB (+), increase in pBOO	Aydin et al. (12)
KCa1.1 β1 (KCNMB1)	mRNA	Rat UBSM tissue with mucosa	Quantitative RT-PCR (+), increase in pBOO	Kita et al. (84)
KCa1.1 β1 (KCNMB1)	mRNA	Rat UBSM cells	RT-PCR (+)	Chen and Petkov (31)
KCa1.1 β1 (KCNMB1)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Chen and Petkov (31); Layne et al. (94)
KCa1.1 β1 (KCNMB1)	Protein	Mouse UBSM tissue	LacZ expression, lacZ was targeted to KCNMB1 for knockout generation	Petkov et al. (131)
KCa1.1 β1 (KCNMB1)	mRNA	Mouse UBSM cells	RT-PCR (+)	Chen and Petkov (31)
KCa1.1 β1 (KCNMB1)	Protein	Rabbit UBSM tissue	WB (+), decrease in pBOO (semiquantitative)	Chang et al. (27)
KCa1.1 β2 (KCNMB2)	mRNA	Human UBSM tissue	RT-PCR (−)	Hristov et al. (67)
KCa1.1 β2 (KCNMB2)	mRNA	Human UBSM cells	RT-PCR (−)	Hristov et al. (67)
KCa1.1 β2 (KCNMB2)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low expression and unchanged in pBOO	Chen and Petkov (31); Kita et al. (84)
KCa1.1 β2 (KCNMB2)	mRNA	Rat UBSM tissue with mucosa	Quantitative RT-PCR (+), decrease/no change in pBOO	Kita et al. (84)
KCa1.1 β2 (KCNMB2)	mRNA	Rat UBSM cells	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β2 (KCNMB2)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Chen and Petkov (31)
KCa1.1 β2 (KCNMB2)	mRNA	Mouse UBSM cells	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β3 (KCNMB3)	mRNA	Human UBSM tissue	RT-PCR (−)	Hristov et al. (67)
KCa1.1 β3 (KCNMB3)	mRNA	Human UBSM cells	RT-PCR (−)	Hristov et al. (67)
KCa1.1 β3 (KCNMB3)	mRNA	Rat UBSM tissue	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β3 (KCNMB3)	mRNA	Rat UBSM cells	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β3 (KCNMB3)	mRNA	Mouse UBSM tissue	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β3 (KCNMB3)	mRNA	Mouse UBSM cells	RT-PCR (−)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), unchanged in NDO	Hristov et al. (67); Hristov et al. (65)
KCa1.1 β4 (KCNMB4)	Protein	Human UBSM tissue	WB (+)	Hristov et al. (67)
KCa1.1 β4 (KCNMB4)	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (67)
KCa1.1 β4 (KCNMB4)	Protein	Human UBSM cells	ICC (+)	Hristov et al. (67)
KCa1.1 β4 (KCNMB4)	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+), decrease in pBOO	Kita et al. (84)
KCa1.1 β4 (KCNMB4)	Protein	Rat UBSM tissue	WB (+)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	mRNA	Rat UBSM tissue with mucosa	Quantitative RT-PCR (+), decrease in pBOO	Kita et al. (84)
KCa1.1 β4 (KCNMB4)	mRNA	Rat UBSM cells	RT-PCR (+)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	Protein	Rat UBSM cells	ICC (+)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	Protein	Mouse UBSM tissue	WB (+)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	mRNA	Mouse UBSM cells	RT-PCR (+)	Chen and Petkov (31)
KCa1.1 β4 (KCNMB4)	Protein	Mouse UBSM cells	ICC (+)	Chen and Petkov (31)
KCa2.1 (KCNN1)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), high relative expression; quantitative RT-PCR (−)	Afeli et al. (5); Chen et al. (32)
KCa2.1 (KCNN1)	mRNA	Human UBSM cells	RT-PCR (−); quantitative RT-PCR (+/−), low/negligible relative expression	Afeli et al. (5)
KCa2.1 (KCNN1)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Parajuli et al. (127)
KCa2.1 (KCNN1)	Protein	Guinea pig UBSM tissue	WB (+)	Parajuli et al. (127)
KCa2.1 (KCNN1)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Parajuli et al. (127)
KCa2.1 (KCNN1)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), unchanged in pBOO	Parajuli et al. (123); Kita et al. (84)
KCa2.1 (KCNN1)	mRNA	Rat UBSM tissue with mucosa	RT-PCR (+); quantitative RT-PCR (+), unchanged in pBOO	Kim and Yang (83); Kita et al. (84)
KCa2.1 (KCNN1)	mRNA	Rat UBSM cells	RT-PCR (−)	Parajuli et al. (123)
KCa2.1 (KCNN1)	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+)	Thorneloe et al. (167)
KCa2.1 (KCNN1)	mRNA	Mouse UBSM cells	Quantitative RT-PCR (−)	Lee et al. (97)
KCa2.2 (KCNN2)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low relative expression	Afeli et al. (5); Chen et al. (32)
KCa2.2 (KCNN2)	mRNA	Human UBSM cells	RT-PCR (−); quantitative RT-PCR (+/−), low/negligible relative expression	Afeli et al. (5)
KCa2.2 (KCNN2)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Parajuli et al. (127)
KCa2.2 (KCNN2)	Protein	Guinea pig UBSM tissue	WB (+)	Parajuli et al. (127)
KCa2.2 (KCNN2)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Parajuli et al. (127)
KCa2.2 (KCNN2)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), increase in pBOO; RT-PCR(+), decrease in pBOO (semiquantitative)	Parajuli et al. (123); Kita et al. (84); Li et al. (100)
KCa2.2 (KCNN2)	mRNA	Rat UBSM tissue with mucosa	RT-PCR (+); quantitative RT-PCR (+), unchanged in pBOO	Kim and Yang (83); Kita et al. (84)
KCa2.2 (KCNN2)	Protein	Rat UBSM tissue with mucosa	WB (+)	Kim and Yang (83)
KCa2.2 (KCNN2)	mRNA	Rat UBSM cells	RT-PCR (−)	Parajuli et al. (123)
KCa2.2 (KCNN2)	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+)	Thorneloe et al. (167)
KCa2.2 (KCNN2)	mRNA	Mouse UBSM tissue with mucosa	RT-PCR (−)	Ohya et al. (118)
KCa2.2 (KCNN2)	Protein	Mouse UBSM tissue	LacZ expression (+), lacZ targeted to KCNN2 for knockout generation	Thorneloe et al. (167)
KCa2.2 (KCNN2)	mRNA	Mouse UBSM cells	Quantitative RT-PCR (+/−), low/negligible relative expression	Lee et al. (97)
KCa2.3 (KCNN3)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), high relative expression; quantitative RT-PCR (+), low relative expression	Afeli et al. (5); Chen et al. (32)
KCa2.3 (KCNN3)	Protein	Human UBSM tissue	WB (+); IHC (+) in UBSM cells	Afeli et al. (5)
KCa2.3 (KCNN3)	mRNA	Human UBSM cells	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Afeli et al. (5)
KCa2.3 (KCNN3)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Parajuli et al. (127)
KCa2.3 (KCNN3)	Protein	Guinea pig UBSM tissue	WB (+)	Parajuli et al. (127)
KCa2.3 (KCNN3)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Parajuli et al. (127)
KCa2.3 (KCNN3)	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), increase in pBOO; RT-PCR (+), decrease in pBOO (semiquantitative)	Parajuli et al. (123); Kita et al. (84); Li et al. (100)
KCa2.3 (KCNN3)	mRNA	Rat UBSM tissue with mucosa	RT-PCR (+); quantitative RT-PCR (+), increase in pBOO	Kim and Yang (83); Kita et al. (84)
KCa2.3 (KCNN3)	Protein	Rat UBSM tissue with mucosa	WB (+)	Kim and Yang (83)
KCa2.3 (KCNN3)	mRNA	Rat UBSM cells	RT-PCR (+)	Parajuli et al. (123)
KCa2.3 (KCNN3)	Protein	Rat UBSM cells	ICC (+)	Parajuli et al. (123)
KCa2.3 (KCNN3)	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+)	Thorneloe et al. (167)
KCa2.3 (KCNN3)	mRNA	Mouse UBSM tissue with mucosa	RT-PCR (−)	Ohya et al. (118)
KCa2.3 (KCNN3)	Protein	Mouse UBSM tissue	IHC (+) in UBSM cells; IHC (−) in UBSM cells	Herrera et al. (63); Lee et al. (97)
KCa2.3 (KCNN3)	mRNA	Mouse UBSM cells	Quantitative RT-PCR (+), moderate relative expression	Lee et al. (97)
KCa3.1 (KCNN4)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low relative expression	Afeli et al. (5); Chen et al. (32)
KCa3.1 (KCNN4)	Protein	Human UBSM tissue	WB (−); IHC (−) in UBSM cells	Afeli et al. (5)
KCa3.1 (KCNN4)	mRNA	Human UBSM cells	RT-PCR (−); quantitative RT-PCR (+/−), low/negligible relative expression	Afeli et al. (5)
KCa3.1 (KCNN4)	mRNA	Guinea pig UBSM tissue	RT-PCR (+)	Parajuli et al. (127)
KCa3.1 (KCNN4)	Protein	Guinea pig UBSM tissue	WB (+)	Parajuli et al. (127)
KCa3.1 (KCNN4)	mRNA	Guinea pig UBSM cells	RT-PCR (+)	Parajuli et al. (127)
KCa3.1 (KCNN4)	mRNA	Rat UBSM tissue	RT-PCR (+)	Parajuli et al. (123)
KCa3.1 (KCNN4)	mRNA	Rat UBSM tissue with mucosa	RT-PCR (+)	Kim and Yang (83)
KCa3.1 (KCNN4)	Protein	Rat UBSM tissue with mucosa	WB (+)	Kim and Yang (83)
KCa3.1 (KCNN4)	mRNA	Rat UBSM cells	RT-PCR (−)	Parajuli et al. (123)
KCa3.1 (KCNN4)	mRNA	Mouse UBSM tissue with mucosa	RT-PCR (+)	Ohya et al. (118)
KCa3.1 (KCNN4)	mRNA	Mouse UBSM cells	Quantitative RT-PCR (−)	Lee et al. (97)

K_Ca, Ca²⁺-activated K⁺; UBSM, urinary bladder smooth muscle; NDO, neurogenic detrusor overactivity; BPH, benign prostatic hyperplasia; DO, detrusor overactivity; WB, Western blot; ICC, immunocytochemistry; IHC, immunohistochemistry; pBOO, partial bladder outflow obstruction; (+), positive detection; (−), negative detection; (+/−), very low/borderline detection.

The K_Ca1.1 channel pore-forming α-subunit, encoded by a single KCNMA gene, can be alternatively processed to generate a number of splice variants possessing differential regulatory properties (29). However, very little is known about K_Ca1.1 splice variants in UBSM. Moreover, associations of regulatory β-subunits with the K_Ca1.1 channels have been established. In addition to the β₁-subunit, which is the primary auxiliary K_Ca1.1 channel subunit in UBSM (131), similar to other smooth muscle cell types (e.g., vascular, intestinal, and uterine), the β₄-subunit has also been detected at mRNA and protein levels in UBSM cells of various species (31, 67, 84). Consistently, K_Ca1.1 channel β₂- and β₃-subunits have not been found in UBSM cells (31, 67). The exact subunit composition, including splice variant expression, of native human (and animal) UBSM K_Ca1.1 channels remains to be determined. The presence of K_Ca1.1 channel β₁- and β₄-subunits increases the apparent Ca²⁺ and voltage sensitivities of K_Ca1.1 channels as well as current kinetics (131). If UBSM cells express multiple channel combinations (α/α, α/β₁, and α/β₄), then a range of voltage activation profiles for K_Ca1.1 channels is expected. Accordingly, single channel recordings in excised patches showed a wide range of voltage activation constants for individual human UBSM K_Ca1.1 channels, thus supporting the existence of multiple combinations (104).

Studies on genetic knockout (KO) models revealed that native K_Ca1.1 channels contain at least α-β₁ complexes regulating urinary bladder function. Targeted deletion of the K_Ca1.1 channel pore-forming α-subunit or regulatory β₁-subunit resulted in increased UBSM excitability and contractility in mouse models (21, 106, 131). Data from global and smooth muscle-specific K_Ca1.1 channel α-subunit KO mice indicated that loss of the K_Ca1.1 channel α-subunit dramatically increased spontaneous and nerve-evoked UBSM contractions, and these transgenic mice displayed an elevated urination frequency (21, 106, 156, 168, 175). While the role of the β₁-subunit in UBSM function has been well established, the function of the more recently discovered β₄-subunit in UBSM is less clear (31, 67, 84). The K_Ca1.1 β₄-subunit may have a role in UBSM pathophysiology (65, 84).

K_Ca1.1 channels are blocked with high potency and relative selectivity by two peptides, iberiotoxin and charybdotoxin, although the latter also potently inhibits K_v1.3 and K_Ca3.1 (IK) channels (128–130). Moreover, all known K_Ca1.1 channel splice variants are effectively blocked by paxilline (87), thus making paxilline ideal for UBSM K_Ca1.1 channel functional studies. K_Ca1.1 channels are also inhibited nonselectively by tetraethylammonium, but with low potency (21, 62, 129). Pharmacological blockade of K_Ca1.1 channels affected UBSM cell excitability, specifically by causing increases in the amplitude and duration of spontaneous action potentials. This indicates that the repolarization phase of the UBSM action potential is mediated by K_Ca1.1 channel activity (52, 53, 55, 57). Although some studies have failed to report UBSM resting membrane potential depolarization upon K_Ca1.1 channel pharmacological inhibition (52, 53, 55), it is now well documented that blocking K_Ca1.1 channels depolarizes the resting membrane potential in both isolated UBSM cells and intact tissues (57, 67, 69, 71, 177, 178, 180, 183). Moreover, genetic deletion of the K_Ca1.1 channel α-subunit caused a sustained membrane depolarization in UBSM (21, 156). The effects of K_Ca1.1 channel blockers on UBSM cell excitability have translated into enhancements in muscle contractility. In UBSM of various animal species and humans, pharmacological inhibition of K_Ca1.1 channels (e.g., iberiotoxin or paxilline) increased the amplitude, duration, and force of the spontaneous phasic contractions as well as UBSM tone (21, 35, 60, 65, 67, 69, 71, 77, 115, 131, 177, 178, 183). In contrast, iberiotoxin displayed no effect on phasic and tonic contractions of UBSM strips from mice that lacked the K_Ca1.1 channel α-subunit (21). Pharmacological inhibition or genetic deletion of K_Ca1.1 channels enhanced nerve-evoked contractions in UBSM strips from different species (3, 4, 59, 67, 91, 156, 168, 175). On the other hand, K_Ca1.1 channel activators such as NS1619, NS8, and NS11021 increased K_Ca1.1 channel open probability and whole cell K_Ca1.1 currents and caused membrane hyperpolarization as well as relaxation of UBSM (27, 71, 74, 77, 93, 102, 115, 147, 155).

Ca²⁺ is a critical K_Ca1.1 channel regulator (128, 129). In UBSM, there are two major Ca²⁺ sources for K_Ca1.1 channel activation: 1) Ca²⁺ entry through Ca_v1.2 channels and 2) Ca²⁺ release from ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) (61, 62, 67, 69, 134). Other sources for intracellular Ca²⁺ in UBSM cells might also potentially originate via Ca_v3 channels, inositol 1,4,5-trisphosphate (IP₃) receptors from the SR, Na⁺/Ca²⁺ exchangers, and SR store-operated channels, although these Ca²⁺ sources have not been well characterized or established for K_Ca1.1 channel activation. K_Ca1.1 channels are under the local control of so-called “Ca²⁺ sparks.” Ca²⁺ spark events are caused by spontaneous highly localized and transient Ca²⁺ releases via RyRs that subsequently trigger transient K_Ca1.1/BK currents (TBKCs), originally known as spontaneous transient outward currents (STOCs), without affecting the global intracellular Ca²⁺ concentration (61, 116, 134, 180). Ca²⁺ sparks and related TBKCs have been reported in UBSM cells from various species (21, 61, 62, 69, 116, 134, 179, 180, 183), including humans (65, 67, 181). In membrane potential recordings, TBKCs manifest as STHs that contribute to UBSM hyperpolarization (67, 179–183). In agreement, TBKC and STH frequencies correlated with each other in UBSM cells, and both were completely inhibited by K_Ca1.1 channel blockers such as iberiotoxin or paxilline (67, 177, 178, 180, 183).

Bladder function is regulated by parasympathetic nerve fibers that release acetylcholine (ACh), which is the primary excitatory neurotransmitter released from nerve varicosities. The released ACh activates M receptors in UBSM cells, initiating physiological phasic contractions that facilitate voiding (9). Recent evidence supports the involvement of K_Ca1.1/BK channels downstream of M receptor activation in UBSM cells. In rat UBSM cells, activation of M3 receptors with carbachol caused an initial appearance of large transient outward K_Ca1.1 currents (TBKCs) followed by sustained inhibition of spontaneous TBKCs (125). In the current-clamp mode (current = 0), these effects translated into reductions of STHs and depolarization of UBSM cell resting membrane potentials (125). These large carbachol-induced TBKCs were attributed to IP₃ receptor-induced SR Ca²⁺ release as the IP₃ receptor inhibitor xestospongin-C completely blocked their generation (125). Furthermore, depletion of SR Ca²⁺ upon sustained activation of M3 receptors reduced Ca²⁺ spark activity, inhibited spontaneous TBKCs, and depolarized UBSM cell membrane potentials, causing an increase in L-type Ca_v channel activity and enhancement of UBSM contractility. This mechanism of M receptor-mediated inhibition of K_Ca1.1 channels has now also been confirmed to operate in human UBSM cells (122). Furthermore, M3 receptor intracellular signaling also involves the activation of protein kinase C, which has been shown to regulate K_Ca1.1 channels in UBSM (72, 75). Specifically, phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibited K_Ca1.1 channel activity indirectly via a Ca²⁺-dependent mechanism involving the attenuation of Ca²⁺ release through RyRs while also increasing the global intracellular Ca²⁺ necessary to activate UBSM contraction (72).

In UBSM, stimulation of β-ARs can increase intracellular cAMP concentrations leading to the activation of protein kinase A (PKA), which, in turn, phosphorylates specific proteins resulting in decreased UBSM excitability and contractility (9, 21, 54, 109). Upon pharmacological activation of β-ARs, K_Ca1.1 channel activity increased, and this effect promoted relaxation of UBSM (21, 69, 134). Additionally, PKA stimulation activated Ca²⁺ sparks in guinea pig UBSM (88, 134, 180). The critical role of K_Ca1.1 channels in β-AR/PKA-mediated UBSM relaxation was supported by the fact that K_Ca1.1 channel genetic deletion in transgenic mouse models led to a compensatory upregulation of the β-AR/PKA pathway (21, 156).

UBSM expresses all three known β-AR subtypes (β₁−β₃) with profound species-dependent expression patterns (9). β₃-ARs appear to be the most physiologically relevant in human UBSM (184). In rat UBSM, experimental findings provided evidence that β₃-ARs and K_Ca1.1 channels functionally couple to promote UBSM relaxation by a complex Ca²⁺-dependent mechanism (69). In rat and human UBSM, β₃-AR agonists effectively inhibited both spontaneous and nerve-evoked UBSM contractions. A role for K_Ca1.1 channels in β₃-AR agonist-mediated relaxation has been concluded by reports of attenuated contractile inhibitory effects to β₃-AR agonism in the presence of a K_Ca1.1 channel inhibitor (e.g., iberiotoxin) (3, 4, 69, 164). In contrast, although studies have reported β₃-AR expression at the mRNA level in guinea pig UBSM, β₃-ARs appeared to have a negligible role, if any, in UBSM contractility without affecting cell excitability (1). Collectively, selective β₃-AR agonists are effective in controlling UBSM function. Indeed, the newly approved selective β₃-AR agonist mirabegron exhibits favorable clinical efficacy in the management of OAB (28).

In UBSM cells, phosphodiesterases targeting cAMP hydrolysis exhibit constitutive activity (177, 178, 180, 183). Their pharmacological inhibition can lead to a rapid increase in cellular cAMP levels, which, in turn, reinforces PKA signaling pathways. In the case of UBSM K_Ca1.1 channels, phosphodiesterase inhibition led to their activation in a RyR-dependent manner (177, 178, 180, 183). Phosphodiesterase-1 and phosphodiesterase-4 appear to have particular functional roles in UBSM based on pharmacological data. Selective phosphodiesterase-1 inhibition with 8-methoxymethyl-3-isobutyl-1-methylxanthine suppressed human UBSM excitability and contractility via activation of RyRs and an increase in TBKC frequency (181, 183). Selective blockade of phosphodiesterase-4 with rolipram or Ro-20-1724 reduced UBSM contractility by increasing the frequency of Ca²⁺ sparks, TBKCs, and decreasing the global intracellular Ca²⁺ concentration (180).

In addition to the K_Ca1.1 channel regulatory mechanisms described above, various substances, hormones, and endogenous factors can also modulate UBSM K_Ca1.1 channels. Sex hormones such as estrogens and testosterone directly activated K_Ca1.1 channels and induced UBSM relaxation (51, 64, 70, 139). Ethanol increased K_Ca1.1 channel activity by differential mechanisms involving dependence on intracellular Ca²⁺ and the basal level of TBKC frequency in UBSM cells (101). Hydrogen sulfide enhanced UBSM contractility via direct inhibition of UBSM K_Ca1.1 channels (39). Moreover, PGE₂ increased UBSM excitability and contractility by selectively inhibiting K_Ca1.1 channel-dependent TBKCs (126).

The importance of K_Ca1.1 channels in UBSM excitability and contractility suggests that K_Ca1.1 channel defects, alterations, or mutations may cause certain forms of LUTS, including DO or detrusor underactivity. Decreased K_Ca1.1 channel expression has been reported in patients with BPH-induced pBOO and associated DO as well as in rabbits with pBOO-induced DO (27). Genetic ablation of the K_Ca1.1 channel pore-forming α-subunit or regulatory β₁-subunit caused enhanced UBSM contractility (21, 106, 131, 156, 168). Overexpression of the K_Ca1.1 channel α-subunit using gene transfer techniques could eliminate DO caused by pBOO in rats (34), an observation consistent with increased UBSM contractility in KO mice lacking K_Ca1.1 channel subunits (21, 106, 131, 156, 168). Genetic deletion of the K_Ca1.1 channel β₁-subunit, favoring the expression of homomeric K_Ca1.1 α-channels rather than α-β₁ K_Ca1.1 complexes, resulted in decreased single K_Ca1.1 channel open probability, causing DO (131). Furthermore, K_Ca1.1 channel α-subunit mRNA expression, whole cell K_Ca1.1 currents, and TBKCs were all decreased in UBSM from patients with neurogenic DO (NDO) compared with control patients (65). Moreover, expression of the K_Ca1.1 β₁-subunit was unchanged, whereas the β₄-subunit increased, in NDO UBSM, potentially indicating a compensatory mechanism (65). UBSM strips isolated from patient donors with NDO consistently did not respond to the K_Ca1.1 channel blocker iberiotoxin or opener NS1619, indicating K_Ca1.1 channel dysfunction in these patients (115). Collectively, these data show that at least certain types of NDO are associated with decreased UBSM K_Ca1.1 channel expression and function, thus contributing to increased UBSM excitability and contractility.

K_Ca1.1 channel pharmacological activation with selective openers has long been explored as a pharmacological approach for LUTS treatment (24, 35, 71, 74, 77, 91, 93, 147, 155, 172). The K_Ca1.1 channel opener NS1619 significantly increased whole cell K_Ca1.1 current, caused membrane hyperpolarization, decreased the level of global intracellular Ca²⁺, and inhibited myogenic and nerve-evoked contractions in native human UBSM (71). In excised membrane patches from human UBSM cells, NS1619 drastically enhanced single K_Ca1.1 channel open probability by ~10-fold with the primary effect on channel gating (104). Interestingly, NS1619 inhibited UBSM action potentials and at higher concentrations caused hyperpolarization (163). In vivo administration of the K_Ca1.1 channel activator NS8 resulted in increased storage capacity of the bladder and reversed bladder hyperactivity in rats (91). Intravenous administration of NS8 in rats also caused a reduction in micturition frequency and an increase in micturition volume (165). K_Ca1.1 channel openers, at lower doses, may affect bladder function without vascular effects. Indeed, drug discovery efforts have been undertaken to develop more potent and selective K_Ca1.1 channel openers (24, 92, 172). At present, however, they have not been successful beyond early clinical stages. A key limitation is the lack of understanding of how UBSM K_Ca1.1 channels, especially in humans, are different at both the expression (composition) and regulatory levels from those present elsewhere in the body. Additionally, inhibition of K_Ca1.1 channels with a selective inhibitor could stimulate UBSM contractility and could be considered as a potential treatment for detrusor underactivity. This is an intriguing new area of investigation.

UBSM K_Ca1.1 channel gene therapy represents another potential clinical application for the treatment of LUTS. Currently, the K_Ca1.1 α-channel genetic construct (URO-902 by Urovant Sciences, https://urovant.com/pipeline/) applied directly to the urinary bladder is being investigated in clinical phase 2 trial for OAB. In a phase 1 study (144), URO-902 displayed a highly favorable safety profile. Thus, the K_Ca1.1 channel is a promising novel opportunity for therapeutic intervention in human UBSM for the treatment of OAB associated with DO.

UBSM K_Ca3.1 channels.

K_Ca3.1 channels have been shown to be expressed at the mRNA level in mouse UBSM (118). Our group has also found mRNA expression of K_Ca3.1 channels in guinea pig UBSM using single cell RT-PCR and further confirmed K_Ca3.1 channel protein expression with Western blot analysis (127). K_Ca3.1 channel protein, however, was not found in rat and guinea pig UBSM using immunostaining (5, 123). Human UBSM cells did not express mRNA for K_Ca3.1 channels (5). Studies using charybdotoxin, a K_Ca3.1 channel inhibitor, suggested that this K_Ca channel subtype may have a functional role in mouse UBSM (55, 118); however, this requires further evidence, as charybdotoxin inhibits both K_Ca3.1 and K_Ca1.1 channels (129, 130). Although the K_Ca3.1 channel might be expressed at the mRNA and protein levels, patch-clamp and functional studies with the selective inhibitor TRAM-34 did not confirm a role for K_Ca3.1 channels in guinea pig, rat, or human UBSM excitability and contractility under normal (nondiseased) physiological conditions (5, 123, 127, 154). It is possible that K_Ca3.1 channels may become active under pathophysiological conditions, but this requires further investigation. Genetic KO or knockin approaches to K_Ca3.1 channels in UBSM cells have not yet been used to investigate their roles in urinary bladder function.

UBSM SK/K_Ca2.x channels.

The K_Ca2 channels (Fig. 1) regulated by Ca²⁺ and calmodulin contribute to the UBSM action potential afterhyperpolarization phase (43, 52). The low K_Ca2 channel density in UBSM cells (62), along with their Ca²⁺ sensitivity, makes these channels unique sensors for changes in global intracellular Ca²⁺ levels. Detection methods revealed the presence of K_Ca2 channel subtypes in UBSM cells and tissues of various species at mRNA and protein levels (Table 3). The peptide apamin is pan selective for K_Ca2 channels, as this molecule inhibits all three subtypes (K_Ca2.1–K_Ca2.3). When examined in terms of UBSM contractility, apamin induced enhancement of contractility (5, 35, 43, 59, 60, 62, 63, 77, 123, 127, 154, 167). In UBSM tissue (muscle bundle) using sharp microelectrode recording, apamin converted single action potentials into bursts by attenuating the initial action potential hyperpolarization without affecting the resting membrane potential (52–54). Similarly, in single UBSM cells, apamin per se did not change membrane potential (123, 127). The lack of an apamin effect on the membrane potential examined indicates that, at least under these specific test conditions, K_Ca2 channels were not active in UBSM cells.

Genetic studies have also supported a role for K_Ca2 channels in the regulation of UBSM function. Overexpression of the K_Ca2.3 channel in mice elevated UBSM K_Ca2 currents and also increased bladder capacity and decreased the frequency of UBSM phasic contractions, whereas suppression of K_Ca2.3 channel expression increased the frequency of UBSM phasic contractions in vitro and nonvoiding contractions in vivo (63). In mouse K_Ca2.2 KOs, apamin did not display robust potentiation of spontaneous phasic and electrical field-stimulated contractions in UBSM strips in contrast to the effects observed in wild-type mice (167).

Pharmacological activation of K_Ca2 channels with selective channel openers might be a novel approach to restoring normal UBSM function, when overactive, as shown in vivo and in vitro (112, 121, 123). K_Ca2 channel activation with SKA-31, the most potent and selective K_Ca2/K_Ca3.1 channel activator, reduced the excitability and contractility of guinea pig and human UBSM (127, 154). The K_Ca2/K_Ca3.1 channel openers NS309 and SKA-31 dramatically increased the duration of the afterhyperpolarization and then abolished action potential firing in an apamin-sensitive manner (163). NS309 decreased rat UBSM cell excitability and contractility by targeting the K_Ca2.3 channel, the main K_Ca2 channel subtype in UBSM (123). Collectively, these studies also established that K_Ca2 channels, but not K_Ca3.1 channels, mediate the inhibitory effects on UBSM excitability and contractility (5, 123, 127, 154). While it is obvious that K_Ca2 channels have fundamental roles in regulating UBSM function, knowledge of their regulatory mechanisms in human UBSM remains limited (32, 35, 52, 154). In addition, very little is known about the role of K_Ca2 channels in urinary bladder disease, and this area requires future investigation. Interpretation of the role of K_Ca2 channels in the whole bladder is even more complex given that non-UBSM cells within the bladder wall, such as PDGFRα⁺ interstitial cells, also express K_Ca2 channels (95–97). In addition, given the differential expression profiles of K_Ca2 subtypes among species, observations on animal models may not directly translate to human UBSM (Table 3).

UBSM Inward-Rectifying ATP-Sensitive K⁺ channels (K_ir6-Sulfonylurea Receptors)

In UBSM, K_ATP channels (Fig. 1), known as K_ir6 channels coassembled with sulfonylurea receptor (SUR) subunits to form functional channels (81, 82), have been detected at mRNA and protein levels in UBSM (Table 4). K_ATP channels link the cell metabolic state (intracellular ATP concentration) with membrane potential and can decrease or increase cell excitability accordingly. However, K_ATP channels are neither Ca²⁺ nor voltage regulated, and this profile disconnects them from these two most important physiological cellular regulators. The original recordings of whole cell K_ATP currents were made in the early 1990s in guinea pig UBSM cells (18) followed by determinations at the single channel level in pig and human UBSM cells (81, 82). The K_ATP channel inhibitor glibenclamide (132, 148), at concentrations exhibiting high affinity and potency for K_ATP channels, did not affect UBSM excitability or contractility in the absence of K_ATP channel stimulation, questioning whether or not K_ATP channels contribute to UBSM resting membrane potentials under normal physiological conditions (43, 77, 82). On the contrary, pharmacological activation of K_ATP channels caused membrane hyperpolarization and via closure of Ca_v1.2 channels led to a reduction in UBSM contractility (40, 43, 54, 56, 81, 82, 132, 148). It is of note that only a very small level of hyperpolarization (~2–3 mV) has been shown to be sufficient to prevent the opening of Ca_v1.2 channels by K_ATP agonism as long as the membrane potential is below the threshold needed for the action potentiation initiation. Indeed, only ~1% of the K_ATP channels needed to be activated to inhibit UBSM action potentials and related phasic UBSM contractions (132).

Table 4.

Expression of K_ir, SUR, and K_2P channels in UBSM whole tissues and isolated cells

Type	Detection	Preparation	Method/Finding	Reference(s)
Kir6.1 (KCNJ8)	mRNA	Human UBSM tissue	RT-PCR (+)	Kajioka et al. (82)
Kir6.1 (KCNJ8)	mRNA	Pig UBSM tissue	RT-PCR (+)	Kajioka et al. (81)
Kir6.1 (KCNJ8)	mRNA	Rat UBSM tissue	RT-PCR (+)	Philyppov et al. (135)
Kir6.2 (KCNJ11)	mRNA	Human UBSM tissue	RT-PCR (−)	Kajioka et al. (82)
Kir6.2 (KCNJ11)	mRNA	Pig UBSM tissue	RT-PCR (+)	Kajioka et al. (81)
Kir6.2 (KCNJ11)	mRNA	Cultured guinea pig UBSM cells	RT-PCR (+)	Gopalakrishnan et al. (46)
Kir6.2 (KCNJ11)	mRNA	Rat UBSM tissue	RT-PCR (+)	Philyppov et al. (135)
SUR1 (ABCC8)	mRNA	Human UBSM tissue	RT-PCR (−); quantitative RT-PCR (−)	Kajioka et al. (82)
SUR1 (ABCC8)	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (−)	Kajioka et al. (82)
SUR1 (ABCC8)	Protein	Human UBSM tissue	IHC (+) in UBSM cells	Aishima et al. (6)
SUR1 (ABCC8)	mRNA	Pig UBSM tissue	RT-PCR (−)	Kajioka et al. (81)
SUR1 (ABCC8)	mRNA	Cultured guinea pig UBSM cells	RT-PCR (+)	Gopalakrishnan et al. (46)
SUR1 (ABCC8)	mRNA	Rat UBSM tissue	RT-PCR (−)	Philyppov et al. (135)
SUR2A (ABCC9)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR, (+) trace/very low relative expression	Kajioka et al. (82)
SUR2A (ABCC9)	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (−)	Kajioka et al. (82)
SUR2A (ABCC9)	mRNA	Pig UBSM tissue	RT-PCR (+)	Kajioka et al. (81)
SUR2A (ABCC9)	mRNA	Cultured guinea pig UBSM cells	RT-PCR (−)	Gopalakrishnan et al. (46)
SUR2A (ABCC9)	mRNA	Rat UBSM tissue	RT-PCR (−)	Philyppov et al. (135)
SUR2A (ABCC9)	mRNA	Mouse UBSM tissue (with mucosa)	RT-PCR (+)	Isomoto et al. (79)
SUR2B (ABCC9)	mRNA	Human UBSM tissue	RT-PCR (+); quantitative RT-PCR (+)	Kajioka et al. (82)
SUR2B (ABCC9)	Protein	Human UBSM tissue	IHC (+) in UBSM cells	Aishima et al. (6)
SUR2B (ABCC9)	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (+)	Kajioka et al. (82)
SUR2B (ABCC9)	mRNA	Pig UBSM tissue	RT-PCR (+)	Kajioka et al. (81)
SUR2B (ABCC9)	mRNA	Cultured guinea pig UBSM cells	RT-PCR (+)	Gopalakrishnan et al. (46)
SUR2B (ABCC9)	mRNA	Rat UBSM tissue	RT-PCR (+)	Philyppov et al. (135)
SUR2B (ABCC9)	mRNA	Mouse UBSM tissue (with mucosa)	RT-PCR (+)	Isomoto et al. (79)
K2P2.1/TREK-1 (KCNK2)	mRNA	Human UBSM tissue	RT-PCR (+), decrease in DO (semiquantitative)	Pineda et al. (137)
K2P2.1/TREK-1 (KCNK2)	Protein	Human UBSM tissue	WB (+), decrease in DO; IHC (+) in UBSM cells	Pineda et al. (137)
K2P2.1/TREK-1 (KCNK2)	mRNA	Human UBSM cells	RT-PCR (+)	Baker et al. (14)
K2P2.1/TREK-1 (KCNK2)	Protein	Cultured human UBSM cells	ICC (+)	Pineda et al. (137)
K2P2.1/TREK-1 (KCNK2)	mRNA	Cultured human UBSM cells	RT-PCR (+)	Pineda et al. (137)
K2P2.1/TREK-1 (KCNK2)	mRNA	Monkey UBSM cells	RT-PCR (+)	Baker et al. (14)
K2P2.1/TREK-1 (KCNK2)	Protein	Mouse UBSM tissue	IHC (+) in UBSM cells, decrease in pBOO; WB (+), decrease in pBOO	Baker et al. (13)
K2P2.1/TREK-1 (KCNK2)	Protein	Mouse UBSM (with mucosa)	IHC (+) in UBSM cells; WB (+)	Pineda et al. (136)
K2P2.1/TREK-1 (KCNK2)	mRNA	Mouse UBSM cells	RT-PCR (+)	Baker et al. (14)
K2P3.1/TASK-1 (KCNK3)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Beckett et al. (15)
K2P3.1/TASK-1 (KCNK3)	Protein	Mouse UBSM tissue	WB (+); IHC (+) in UBSM cells	Beckett et al. (15)
K2P3.1/TASK-1 (KCNK3)	Protein	Mouse UBSM cells	ICC (+)	Beckett et al. (15)
K2P5.1/TASK-2 (KCNK5)	mRNA	Mouse UBSM tissue	RT-PCR (+)	Beckett et al. (15)
K2P5.1/TASK-2 (KCNK5)	Protein	Mouse UBSM tissue	WB (+); IHC (+) in UBSM cells	Beckett et al. (15)
K2P5.1/TASK-2 (KCNK5)	Protein	Mouse UBSM cells	ICC (+)	Beckett et al. (15)

K_ir, inward-rectifying K⁺; SUR, sulfonylurea receptor; K_2P, two-pore domain K⁺; TREK, TWIK-related K⁺; TASK, TWIK-related acid-sensitive K⁺; UBSM, urinary bladder smooth muscle; IHC, immunohistochemistry; DO, detrusor overactivity; WB, Western blot; ICC, immunocytochemistry; pBOO, partial bladder outflow obstruction; (+), positive detection; (−), negative detection.

Intriguingly, since K_ATP channel density has been determined to be much higher in UBSM compared with other tissues, including vascular (81, 132, 148), one way to achieve bladder-selective effects may be to apply lower concentrations of K_ATP channel openers without affecting other tissues. Human UBSM cell K_ATP channels have also been reported to primarily contain a bladder-specific splice variant, called SUR2B-exon17(−)-K_ir6.2, which displays a different pharmacological profile compared with SUR2B-exon17(+)-K_ir6.2, preferentially expressed elsewhere (145). Moreover, even though K_ATP openers advanced to early clinical stages for OAB management (e.g., ZD6169 and ZD0947), they were ultimately terminated during development either due to lack of efficacy over placebo or the presence of side effects (25).

UBSM K_2P Channels

Studies have reported that K_2P channels (Fig. 1), also known as “leak K⁺ channels,” have functional roles in UBSM under normal and pathological conditions (13–15). K_2P channels appear to participate in stabilizing UBSM cell resting membrane potential (15). As shown in Table 4, mouse UBSM cells and tissues were found to express K_2P, stretch-dependent K⁺ channels of the K_2P2.1 or TWIK-related K⁺ channel 1 (TREK-1) subtype (14). They have also been identified in human UBSM cells, cultured human UBSM cells, monkey UBSM cells, and rat UBSM cells and tissues (Table 4) (13, 14, 166). BL-1249, a K_2P2.1 channel opener, produced membrane hyperpolarization in cultured human UBSM cells, inhibited 30 mM KCl-induced UBSM contractions, and decreased frequency of isovolumic contractions in vivo in rats without affecting blood pressure (166). Conversely, methionine, a K_2P2.1 channel inhibitor, increased contractility in UBSM tissue strips and enhanced excitability (14). Mice lacking K_2P2.1 channels displayed altered UBSM contractility and voiding patterns (136). DO caused by pBOO in mice was linked to reduced expression of K_2P2.1 channels; therefore, this subtype may have a physiological role in suppressing UBSM excitability and contractility in response to stretch during bladder filling and thus may contribute to urinary bladder disease.

Moreover, two other members of the K_2P channel family were found in UBSM. They are K_2P3.1 or TWIK-related acid-sensitive K⁺ type 1 channels and K_2P5.1 or TWIK-related acid-sensitive K⁺ type 2 channels. In addition to the mRNA and protein detections (Table 4), observations of UBSM cell whole cell currents displaying acid (pH) and pharmacological sensitivity to lidocaine, a nonselective K_2P3.1/K_2P5.1 channel blocker, functionally supported their existence (15). However, very little is known about their role in UBSM.

UBSM TRP CATION CHANNELS

TRP channels are nonselective cation channels. As a large superfamily of 28 members, these channels have been classified into the following 7 related subfamilies: the TRP canonical (TRPC), TRP vanilloid (TRPV), TRP melastatin (TRPM), TRP polycystin (TRPP), TRP mucolipin (TRPML), TRP no mechanopotential (TRPN), and TRP ankyrin (TRPA) (114). They respond to physical and chemical stimuli such as temperature, pH, stretch, and light as well as intracellular stimuli such as Ca²⁺. They constitute a fundamental mechanism by which cells respond to changes in the environment (114). TRP channels have been reported in bladder urothelium and nerves, but elucidation of their role in UBSM is just now emerging (41, 150) (Fig. 1).

UBSM TRPM4 Channels

Initial in-house microarray data indicated that in human UBSM, TRPM4 channel mRNA expression was much higher than that for other TRP channels (G. Petkov, unpublished observations). This observation focused attention toward this particular TRP channel member. The TRPM4 channel is unique because it is a Ca²⁺-activated cation channel that is highly selective for monovalent cations, such as Na⁺ and K⁺, but impermeable to anions and divalent cations including Ca²⁺ (49, 113). TRPM4 channels exhibit voltage dependency, have a single channel conductance of ~25 pS, and display high Ca²⁺ sensitivity (113). TRPM4 channels are present in UBSM cells and tissues of various species, including humans, rats, guinea pigs, and mice (Table 5) (73, 90, 124, 151, 152). 9-Phenanthrol, a commonly used TRPM4 channel inhibitor (50), has been shown to reduce UBSM excitability. Specifically, 9-phenanthrol hyperpolarized UBSM cell membrane potential and attenuated voltage-step cation currents (73, 103, 105, 124, 151, 152). Furthermore, 9-phenanthrol reduced spontaneous and evoked phasic contractions (20 mM KCl and carbachol) and electrical field-stimulated contractions in UBSM of various species (73, 124, 151, 152). These data suggest that TRPM4 channels have a prominent physiological role in the regulation of UBSM function in humans and animals (73, 124, 151, 152).

Table 5.

Expression of TRP and Cl⁻ channels in UBSM tissue and isolated cells

Type	Detection	Preparation	Method/Finding	Reference(s)
TRPC1	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), high relative expression	Griffin et al. (47); Griffin et al. (48)
TRPC1	mRNA	Mouse UBSM cells	RT-PCR (−)	Griffin et al. (47)
TRPC2	mRNA	Mouse UBSM cells	RT-PCR (+)	Lee et al. (96)
TRPC3	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low/intermediate relative expression	Griffin et al. (47); Griffin et al. (48)
TRPC3	mRNA	Mouse UBSM cells	RT-PCR (−)	Griffin et al. (47)
TRPC4	mRNA	Mouse UBSM tissue	RT-PCR (+)	Griffin et al. (47)
TRPC4	mRNA	Mouse UBSM cells	RT-PCR (+)	Griffin et al. (47)
TRPC4	Protein	Mouse UBSM cells	ICC (+)	Griffin et al. (48)
TRPC5	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), very low relative expression	Griffin et al. (47); Griffin et al. (48)
TRPC5	mRNA	Mouse UBSM cells	RT-PCR (−)	Griffin et al. (47)
TRPC6	mRNA	Human UBSM tissue	RT-PCR (+), increased expression in OAB/BPH (semiquantitative)	Chang et al. (26)
TRPC6	Protein	Human UBSM tissue	WB (+), increased expression in OAB/BPH (semiquantitative); IHC (+) in UBSM cells, increased apparent expression in OAB/BPH	Chang et al. (26)
TRPC6	mRNA	Cultured human UBSM cells	Quantitative RT-PCR (+), pooled cells	Chang et al. (26)
TRPC6	Protein	Cultured human UBSM cells	ICC (+); WB (+), pooled cells	Chang et al. (26)
TRPC6	mRNA	Rat UBSM tissue	RT-PCR (+), increased expression in pBOO (semiquantitative)	Chang et al. (26)
TRPC6	Protein	Rat UBSM tissue	WB (+), increased expression in pBOO (semiquantitative); IHC (+) in UBSM cells, increased apparent expression in pBOO	Chang et al. (26)
TRPC6	mRNA	Mouse UBSM tissue	RT-PCR (+); quantitative RT-PCR (+), low/intermediate relative expression	Griffin et al. (47); Griffin et al. (48)
TRPC6	mRNA	Mouse UBSM cells	RT-PCR (+)	Griffin et al. (47); Lee et al. (96)
TRPC7	mRNA	Mouse UBSM tissue	RT-PCR (−)	Griffin et al. (47)
TRPC7	mRNA	Mouse UBSM cells	RT-PCR (−)	Griffin et al. (47)
TRPV2	mRNA	Mouse UBSM cells	RT-PCR (+)	Lee et al. (96)
TRPV4	mRNA	Mouse UBSM tissue	Quantitative RT-PCR (+)	Thorneloe et al. (170)
TRPV4	mRNA	Mouse UBSM (with mucosa)	RT-PCR (+)	Thorneloe et al. (170)
TRPV4	Protein	Mouse UBSM tissue	WB (+); IHC (+) in UBSM cells	Thorneloe et al. (170); Lee et al. (96)
TRPV4	mRNA	Mouse UBSM cells	RT-PCR (−)	Lee et al. (96)
TRPV4	Protein	Rat UBSM tissue	WB (+), increase in pBOO; IHC (+) in UBSM cells, apparent increase in pBOO	Cho et al. (33)
TRPV4	mRNA	Rat UBSM tissue	Quantitative RT-PCR (+), unaffected/nonsignificant increase in repeated variate stress model	Merrill and Vizzard (107)
TRPV4	Protein	Rat UBSM tissue (with mucosa)	WB (+), increased in repeated variate stress model	Merrill and Vizzard (107)
TRPV4	Protein	Rat UBSM tissue	WB (+), unaffected/nonsignificant increase in repeated variate stress model	Merrill and Vizzard (107)
TRPV4	Protein	Guinea pig UBSM tissue	IHC (+) in UBSM cells; WB (+) increase with aging	Isogai et al. (78); Roberts et al. (142)
TRPM4	mRNA	Human UBSM tissue	RT-PCR (+)	Hristov et al. (73)
TRPM4	Protein	Human UBSM tissue	WB (+); IHC (+) in UBSM cells	Hristov et al. (73)
TRPM4	mRNA	Human UBSM cells	RT-PCR (+)	Hristov et al. (73)
TRPM4	Protein	Human UBSM cells	ICC (+)	Hristov et al. (73)
TRPM4	mRNA	Rat UBSM tissue	RT-PCR (+); quantitative RT-PCR (+)	Smith et al. (152); Parajuli et al. (124)
TRPM4	Protein	Rat UBSM tissue	WB (+); IHC (+) in UBSM cells	Smith et al. (152)
TRPM4	mRNA	Rat UBSM cells	RT-PCR (+)	Smith et al. (152)
TRPM4	Protein	Rat UBSM cells	ICC (+)	Smith et al. (152)
TRPM4	Protein	Guinea pig UBSM tissue	WB (+)	Smith et al. (151)
TRPM4	Protein	Guinea pig UBSM cells	ICC (+)	Smith et al. (151)
TRPM4	mRNA	Mouse UBSM tissue	RT-PCR (+)	Kullmann et al. (90)
TRPM4	Protein	Mouse UBSM tissue	IHC (+) in UBSM cells; WB (+) increase after spinal cord injury	Kullmann et al. (90)
TRPM7	mRNA	Mouse UBSM cells	RT-PCR (+)	Lee et al. (96)
Ano1/TMEM16A	Protein	Human UBSM tissue	IHC (−) in UBSM cells	Gevaert et al. (45)
Ano1/TMEM16A	Protein	Guinea pig UBSM tissue	IHC (−) in UBSM cells	Gevaert et al. (45)
Ano1/TMEM16A	mRNA	Rat UBSM tissue	RT-PCR (+)	Bijos et al. (17)
Ano1/TMEM16A	Protein	Rat UBSM tissue	IHC (−) in UBSM cells; IHC (+) in UBSM cells (and interstitial cells)	Gevaert et al. (45); Bijos et al. (17)
Ano1/TMEM16A	Protein	Mouse UBSM tissue	IHC (−) in UBSM cells	Gevaert et al. (45)
CLCA/CLca5	mRNA	Rat UBSM tissue	RT-PCR (+), increased with pBOO (semiquantitative)	Li et al. (100)
CLCA4	mRNA	Mouse UBSM tissue	RT-PCR (+)	Elble et al. (38)

TRPC, transient receptor potential (TRP) canonical; TRPV, TRP vanilloid; TRPM, TRP melastatin; CLCA, Cl⁻ channel accessory or Ca²⁺-activated Cl⁻ channel regulator; UBSM, urinary bladder smooth muscle; ICC, immunocytochemistry; OAB, overactive bladder; BPH, benign prostatic hyperplasia; WB, Western blot; IHC, immunohistochemistry; pBOO, partial bladder outflow obstruction; (+), positive detection; (−), negative detection.

Since TRPM4 channels are activated by Ca²⁺, IP₃ receptor-mediated Ca²⁺ release from the SR may represent a Ca²⁺ source for TRPM4 channel activation in UBSM. Indeed, using the in situ proximity ligation assay, TRPM4 channels and IP₃ receptors were found to be expressed within the vicinity of each other (141). Furthermore, the selective IP₃ receptor inhibitor xestospongin-C significantly decreased TRPM4 channel-mediated transient inward cation currents, providing additional support that TRPM4 channels and IP₃ receptors are spatially and functionally coupled in human UBSM (141).

9-Phenanthrol, however, is not ideally selective for TRPM4 channels (23, 149); thus, studies with additional, novel, and more selective TRPM4 channel inhibitors are needed. A recent study compared the effects of glibenclamide, a TRPM4 channel or a TRPM4-SUR complex inhibitor, with those of 9-phenanthrol on guinea pig UBSM excitability and contractility (103). Although glibenclamide attenuated excitability and contractility, it showed weaker effects than 9-phenanthrol, suggesting differential mechanisms of action for the two compounds. Furthermore, diazoxide, a SUR-TRPM4 activator, did not enhance voltage step-induced cation currents in UBSM cells, arguing against the role of SUR-TRPM4 channel complexes in the regulation of UBSM function (under normal/control, nondisease conditions) (103). A significant gap in knowledge exists for the role of TRPM4 channels in regulating UBSM function under pathological conditions. Observation of downregulation of TRPM4 channels in UBSM in a mouse model of DO induced by spinal cord injury suggested abnormal function and expression for TRPM4 channels (90).

UBSM TRPV4 Channels

Other TRP channels may also be involved in the regulation of UBSM function (Fig. 1). TRPV4 channels have been detected in UBSM of humans, guinea pigs, and mice (Table 5). GSK1016790A, a selective TRPV4 channel opener, increased UBSM contractions and caused DO in vivo in wild type but not TRPV4 channel KO mice, suggesting a functional role for TRPV4 channels (170). Another study proposed that in UBSM cells, TRPV4 channels functionally couple with K_Ca1.1 channels and provide Ca²⁺ influx to activate K_Ca1.1 channels, thus limiting UBSM contractility (78). However, additional evidence suggests that, at least in the mouse urinary bladder, PDGFRα⁺ interstitial cells, but not UBSM cells, express TRPV4 channels and provide the main source of Ca²⁺ influx activating K_Ca2.3 channels during bladder filling (95, 96). For UBSM TRPV4 channels, the following key questions remain: 1) are TRPV4 channels expressed at sufficient levels in human and animal UBSM cells (e.g., rat) to have a functional role in UBSM; 2) if present, how do TRPV4 channels regulate the function of normal (nondiseased) UBSM; 3) do TRPV4 channels form predominantly heteromeric or homomeric complexes in UBSM; 4) how are they affected by or contribute to urinary bladder disease; and 5) can TRPV4 channels be considered as a therapeutic option for LUTS? At present, only very preliminary evidence is available for the role of TRPV4 in urinary bladder dysfunction. In rat UBSM under pBOO and also mouse whole urinary bladder (UBSM + mucosa) following a repeated variable stress paradigm, TRPV4 channel expression increased (33, 107). Interestingly, TRPV4 channel expression appears to be age dependent. The UBSM (and urothelium) of older guinea pigs expressed higher levels of TRPV4 channel proteins than those of younger animals (142).

Other UBSM TRP Channels

In addition, TRPV2 and TRPC members (TRPC4 and TRPC6) have been detected in UBSM (Fig. 1 and Table 5). The knowledge about their roles and significance in UBSM is limited. TRPC4 channels appear to be important for M receptor-mediated contractions (47, 48). In mice, TRPC4 genetic KO and ML204, a TRPC4 channel inhibitor, attenuated both M receptor-induced UBSM contractions and Ca²⁺ transients (47, 48). In non-UBSM (e.g., vascular) myocytes, TRPC6 channels participate in G protein-coupled receptor signaling pathways that contribute to intracellular Ca²⁺ dynamics, and they may play a similar role in UBSM (37). Indeed, knockdown of TRPC6 channel expression via siRNA reduced extracellular Ca²⁺ entry following intracellular Ca²⁺ store depletion in cultured rat UBSM cells (26). Findings of increased TRPC6 channel expression in UBSM of patients with OAB due to BPH and in pBOO rats suggest a potential role of TRPC6 channels in urinary bladder disease (26). Thus, additional studies are needed on these TRP members in UBSM.

UBSM Cl⁻ CHANNELS

The expression and function of Cl⁻ channels (Fig. 1), including Ca²⁺-activated Cl⁻ (Cl_Ca) channels, in UBSM is largely unexplored. The ion flux via Cl⁻ channels is often assumed to be passive since Cl⁻ is not usually at its equilibrium potential at rest. Cl⁻ channels may be functional in UBSM and potentially modulate membrane potential, where they may be excitatory or inhibitory depending on the driving force (Fig. 1) (129).

UBSM Cl_Ca Channels

Protein and mRNA expression of Ano1/TMEM16A Cl_Ca channels have been reported in UBSM and interstitial UBSM cells of the juvenile rat urinary bladder supported by UBSM contractility studies (17). The functional studies revealed reductions of spontaneous UBSM phasic contractions with the nonselective Cl⁻ channel inhibitors niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (17). Another study reported Ano1/TMEM16A expression in mouse bladder interstitial cells but argued against their presence in UBSM cells (190). More recently, Ano1/TMEM16A proteins were not detected in any cell (including myocytes) with specific immunoreactivity in UBSM tissue sections from human, monkey, mouse, and rat urinary bladders using the same experimental immunostaining conditions that showed a robust detection for Ano1/TMEM16A in interstitial cells of Cajal in the gut (45). Thus, it is evident that the literature is controversial and that the expression and function of Ano1/TMEM16A Cl⁻ channels in UBSM require further investigation. Moreover, no information is available in the literature about other, non-Ano1/TMEM16A Cl_Ca channels in UBSM.

Members of the Cl⁻ channel accessory or Cl_Ca channel regulator (CLCA) family have been detected in UBSM. Although originally CLCA members were considered as integral subunits of Cl_Ca channels, now they are recognized as a new family of self-cleaving metalloproteases that generate secretory peptides exhibiting Cl_Ca channel regulating properties (191). For one such member of the CLCA family (CLca5), increased expression was observed under the condition of DO induced by pBOO in rats (100). Another member, CLCA4, has been detected at the mRNA level in mouse UBSM tissue. The exact roles of CLCA members and how they regulate Cl_Ca channels in UBSM remain to be clarified.

UBSM Ca²⁺-Independent Cl⁻ Channels

Recent evidence from our group suggests that non-Ca_Cl channels can regulate UBSM function (Fig. 1). Conventional whole cell and excised single channel recordings from guinea pig UBSM cells revealed that the Cl⁻ currents measured were not Ca²⁺ or cell volume (hypoosmotic change) sensitive but rather displayed voltage dependency (186, 187). Guinea pig whole cell Cl⁻ currents exhibited a specific anion permeability sequence (Cl⁻ > Br⁻ > I⁻ > methanesulfonate⁻), insensitivity to chlorotoxin (a blocker of small-conductance Cl⁻ channels), and inhibitions by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid (both nonselective inhibitors) (186, 187). Whole cell Cl⁻ currents displayed dependence on extracellular pH, and they were higher and lower at acidic/pH 5 and basic/pH 8.5, respectively (186). Intracellular phosphatidylinositol-4,5-bisphosphate (PIP₂) also regulated whole cell Cl⁻ currents as determined with intracellular PI(4,5)P₂-diC8 (applied via a patch electrode), a metabolically stable PIP₂ analog, which caused potentiation of UBSM Cl⁻ current densities (186). While the currents were not affected by removal of extracellular Ca²⁺ or Mg²⁺, they were regulated by extracellular La³⁺ and Gd³⁺, both of which showed higher currents at 1 mM than at 1 or 50 μM (186, 187). In addition, UBSM cell patches displayed a voltage-dependent Ca²⁺-insensitive single Cl⁻ channel activity of 164 pS measured with the inside-out patch-clamp mode (187). Interestingly, the half-maximum activation constant for this channel activation was −22 mV, i.e., in the physiological range of UBSM cell membrane potentials (−60 to +20 mV) (19, 52, 53). At the peak of the UBSM cell action potential (approximately +10 to +20 mV), this channel would be expected to be fully active.

The molecular identity of native Cl⁻ channels and their roles, including those in urinary bladder pathology in UBSM, await elucidation. It is of note that the pharmacophysiological profile of native UBSM Cl⁻ channels, determined at both single channel and whole cell current levels, does not fit well with any known recombinant Cl⁻ channels (186).

CONCLUSIONS

UBSM relaxation and contraction depend on cell excitability, which is determined by various ion channels described in this review. In general, the openings of Ca_v and TRP channels enhance excitability and contractility while K⁺ channels facilitate a decrease in UBSM cell excitability. Among all known ion channels, Ca_v1.2, K_Ca1.1, K_v2, K_v7, K_ATP, and TRPM4 channels are expressed in UBSM cells/tissues, and they contribute to UBSM function based on experimental evidence summarized in this review. The ultimate integrator of UBSM cell excitability is the Ca_v1.2 channel as its Ca²⁺ influx controls global intracellular Ca²⁺ concentration and subsequently contractility. Other ion channel types, including Ca_v3, K_Ca2, TRPV4, TRPC3/6, various K_2P members, and Cl⁻ channels, have been identified in UBSM based on either expression or functional assessments. Their functional roles remain uncertain due to limits of preliminary findings, controversial results, and lack of sufficient experimental data. However, much remains to be discovered about UBSM ion channel roles and related regulatory mechanisms, especially in human UBSM tissue and cells. Most of our current understanding derives from studies on experimental animal models, but these findings do not always apply directly to humans due to well-documented interspecies differences.

Animal models of urinary bladder dysfunction such as pBOO, spinal cord injury, diabetic phenotype induced, and genetic (e.g., K_Ca1.1 or Ca_v1.2. KO) have identified expression and/or functional deficits for ion channels in UBSM. Furthermore, the very few studies available on UBSM obtained from patient donors with NDO or BPH-DO also suggest the involvement of ion channels, such as K_Ca1.1. Additional and more robust studies, in particular in human UBSM tissues and cells under both normal and pathological conditions, are urgently needed to better our understanding of the role of ion channels in the pathogenesis of LUTS, including OAB/DO. The lack of sufficient data for human UBSM constitutes a key hindrance in understanding the roles of ion channels in determining urinary bladder function in health and disease. Given the critical role of ion channels in UBSM excitability and contractility and their contribution to dysregulation in bladder disease, specific UBSM ion channels may be therapeutically targeted to develop much needed novel drugs to effectively manage LUTS in the future.

GRANTS

This work was supported by National Institutes of Health Grants R01DK106964 and P20DK123971 and by a Van Vleet Endowment to G. V. Petkov.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.M. and G.V.P. drafted manuscript; J.M. and G.V.P. edited and revised manuscript; J.M. and G.V.P. approved final version of manuscript.