Calcium Channels in Vascular Smooth Muscle

Abstract

Calcium (Ca²⁺) plays a central role in excitation, contraction, transcription, and proliferation of vascular smooth muscle cells (VSMs). Precise regulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) is crucial for proper physiological VSM function. Studies over the last several decades have revealed that VSMs express a variety of Ca²⁺-permeable channels that orchestrate a dynamic, yet finely tuned regulation of [Ca²⁺]_i. In this review, we discuss the major Ca²⁺-permeable channels expressed in VSM and their contribution to vascular physiology and pathology.

1. INTRODUCTION

Highly coordinated control of VSM excitability is essential for proper vascular function and regulation of blood flow. Intracellular Ca²⁺ plays a pivotal role in this process. Accordingly, well-orchestrated and distinct signaling pathways allow tight regulation of [Ca²⁺]_i,which, together with differential Ca²⁺ sensitivity of the contractile machinery, provide additional fine-tuning of VSM contractility. Studies over the last several decades have revealed the expression of multiple Ca²⁺-permeable channels in VSM that coordinate a dynamic and precise control of [Ca²⁺]_i, thereby playing a pivotal role in VSM physiology (Fig. 1). Changes in [Ca²⁺]_i are produced by Ca²⁺ influx through voltage-dependent and -independent plasmalemmal Ca²⁺-permeable channels, as well as Ca²⁺ release from intracellular stores. L-type CaV1. channels (LTCCs) have long been considered the primary route of Ca²⁺ entry in VSM. Indeed, Ca²⁺ influx through LTCCs is the principal mediator of myogenic response, which is the intrinsic ability of VSM to contract/relax in response to changes in intraluminal pressure (Bayliss, 1902). Besides LTCC, T-type Ca²⁺ channels (TTCCs) are emerging as important contributors to myogenic tone. LTCCs also play a crucial role in excitation–transcription coupling in VSM (Amberg & Navedo, 2013). Members of the transient receptor potential (TRP) channel family, as well as Ca²⁺ release-activated channels (Orai/STIM), have also been found to contribute to regulation of VSM function. Moreover, Ca²⁺ release from intracellular stores through ryanodine receptors (RyRs) and inositol-1,4,5,-trisphosphate receptors (IP₃Rs) in the sarcoplasmic reticulum (SR) is an important contributor to [Ca²⁺]_i and VSM excitability. RyRs and IP₃Rs are also involved in VSM regulation via their communication with plasmalemmal ion channels. Recently, mitochondria have also been receiving substantial attention for their emerging relevance in VSM Ca²⁺ handling. This review presents an overview of the major Ca²⁺-permeable channels that contribute to VSM Ca²⁺ handling and contractility, and vascular reactivity. More extensive reviews on individual channels can be found elsewhere (Amberg & Navedo, 2013; Earley & Brayden, 2015; Harraz & Welsh, 2013b; McCarron, Olson, Wilson, Sandison, & Chalmers, 2013; Narayanan, Adebiyi, & Jaggar, 2012; Navedo & Amberg, 2013).

Fig. 1.

Schematic representation of the interplay of major Ca²⁺-permeable channels involved in the regulation of VSM [Ca²⁺]_i and contractility. Ca²⁺ influx predominantly through L-type Ca_V1.2 and to some extent T-type Ca_V3.1/3.3 channels promotes VSM contraction. L-type CaV1.2 and T-type Ca_V3.1/3.3 channel activity can be regulated, via membrane potential, by several Ca²⁺-permeable channels serving (1) depolarizing and (2) hyperpolarizing roles, thus modulating the contractile state of VSM. The emerging role of mitochondria Ca²⁺ channels in regulation of VSM Ca²⁺ homeostasis and vascular reactivity is not depicted in this cartoon for simplicity. (+) denotes positive modulation, (−) represents negative modulation, and (?) indicates areas of uncertainty in the pathway.

2. PLASMALEMMAL Ca²⁺-PERMEABLE CHANNELS

2.1 Voltage-Dependent Calcium Channels

Voltage-dependent Ca²⁺ channels (VDCCs) are widely expressed among excitable cells and display a diversity of electrophysiological properties, which allows them to influence many physiological functions (Catterall, 2011). Since their initial discovery in 1953, multiple VDCC subtypes have been characterized (Catterall, 2011). Of particular importance to this review are the LTCCs, which exhibit a large-conductance and long-lasting current with membrane depolarization, and TTCCs with tiny conductance and transient current at negative potentials. N-, P/Q-, and R-type Ca²⁺ channels have also been identified (Catterall, 2011). In the following section, we focus on LTCCs and TTCCs and their contribution to [Ca²⁺]_i in VSM.

2.1.1 L-Type Ca_V1.2 Channels

LTCCs form the fulcrum in Ca²⁺ dynamics of NVSM. Ca²⁺ influx through LTCCs in these cells is a principal mediator of myogenic tone (Fig. 1) (Amberg & Navedo, 2013; Knot & Nelson, 1998; Nelson, Patlak, Worley, & Standen, 1990). The vascular LTCC was first sequenced from rabbit lungs in 1990 (Biel et al., 1990), showing 65% amino acid sequence homology with its skeletal muscle isoform. LTCCs are comprised of pore-forming α_1c and auxiliary β, α₂δ, and γ subunits that modulate channel function. The α_1c, which contains the voltage sensor, the gating apparatus, and the Ca²⁺-permeable pore, is made up of four homologous domains (I, II, III, IV), each of which is composed of six transmembrane segments (S1–S6) and intracellular NH₂- and COOH-termini. The S5 and S6 of each homologous domain form the pore region of the channel. Two glutamate residues at the pore loop determine the Ca²⁺ selectivity. The S1–S4 forms the voltage sensor, which rotates to open the ion pore (Bezanilla, 2008).

The α_1c transcript undergoes extensive alternative spicing, which provides structural and functional diversity in cell-type selective expression patterns. For example, splice variation in rat α_1c exon-1 gives rise to arterial VSM specific α_1c subunit that has cysteine-rich NH₂-terminus (Cheng et al., 2007). This α_1c when coexpressed with only α₂δ demonstrated more negative steady-state activation and deactivation kinetics, smaller whole-cell currents, and decreased plasmalemma incorporation. LTCCs containing a SM-selective 25 amino acid exon 9a in the I–II intracellular linker region exhibit more hyperpolarized window current and are a key regulator of cerebrovascular constriction (Liao et al., 2007; Nystoriak, Murakami, Penar, & Wellman, 2009). Other variations, which exclude exon 33, have window current closer to resting membrane potential and higher sensitivity to blockade by nifedipine (Liao et al., 2007).

The COOH-terminus of α_1c provides regulatory functions such as plasmalemma targeting, retention, and constitutive intracellular recycling of LTCCs (Catterall, 2011). It also contains the calmodulin (CaM)-binding site, which facilitates channel trafficking to the plasmalemma. In rat and human cerebral arteries, the COOH-terminus of α_1c is cleaved, producing a short LTCC and a 50-kDa COOH-terminus fragment (Bannister et al., 2013). This fragment can be detected in the cytosol and the nucleus of VSM and was shown to induce vasodilation by decreasing α_1c expression and shifting the channel voltage dependence of activation to more depolarized potentials. Such findings warrant the need for further in-depth research regarding the role of the α_1c COOH-terminal fragment in blood pressure regulation in physiological and pathological conditions.

In vitro and in vivo studies have established the critical role for α_1c in vascular function. For instance, dihydropyridine antagonists (e.g., nifedipine, isradipine, nicardipine) that selectively inhibit α_1c activity were found to abolish the pressure-induced increase in [Ca²⁺]_i and prevented the development of myogenic tone (Knot & Nelson, 1998). Conversely, dihydropyridine agonists (e.g., Bay K 8644) that specifically stimulate LTCC activity enhance the myogenic response (Hwa & Bevan, 1986). Furthermore, α_1c knockout (SMAKO) mice showed a dramatic drop in myogenic tone and mean arterial pressure (Moosmang et al., 2003). Swelling of VSM may also induce cell contraction through activation of LTCC. Reports from cerebral artery VSM, canine basilar artery VSM, and rat-tail artery VSM confirmed the involvement of LTCC α_1c in vasoconstriction to a hypo-osmotic challenge (Kimura et al., 2000; Welsh, Nelson, Eckman, & Brayden, 2000; Wijetunge & Hughes, 2007).

The β subunit is generally paired in a 1:1 stoichiometry with Ca_V1.2α_1c. The β subunit is made up of two conserved core regions, akin to the Src-homology-3 (SH3) domain and the guanylate kinase domain. Four β sub-units, which are encoded by four different genes with several known splice variants, have been identified. Both biophysical properties and plasmalemmal insertion of Ca_V1.2α_1c can be distinctively regulated by different β isoforms (Catterall, 2011). In VSM, β₃ is the predominant β subunit, and a recent study concluded that it plays a critical role in upregulating LTCC activity and in the development of angiotensin II (ANG II)-induced hypertension (Kharade et al., 2013).

Initially thought to be distinct subunits, the α₂δ subunit exists as a single subunit connected by a disulfide bond (Catterall, 2011). The δ portion is anchored to the plasmalemma, while the glycosylated extracellular α₂ domain interacts with the α_1c (Gurnett, De Waard, &Campbell, 1996). Three different α₂δ isoforms have been identified (α₂δ1– α₂δ3). Heterologous coexpression of different α₂δ isoforms with different α_1c and β subunits produced channels with distinct gating profiles and current densities, highlighting the important regulatory role of α₂δ on channel function (Klugbauer, Lacinova, Marais, Hobom, & Hofmann, 1999). In cerebral VSM, α₂δ is a crucial regulator of LTCC function as illustrated by decreased Ca²⁺ influx via LTCCs and vasodilation following α₂δ1 knockdown (Bannister et al., 2009). Furthermore, increased α₂δ1 mRNA and protein were observed in cerebral VSM from spontaneously hypertensive rats (Bannister et al., 2012). These data, and the β subunit data discussed earlier, suggest that increased α₂δ1 and β3 expression enhances LTCC expression and function, thus contributing to augmented vasoconstriction during hypertension. Therefore, targeting the α₂δ1 and β3 may be a viable therapeutic approach to reverse/ameliorate increased vasoconstriction during hypertension.

Eight γ subunits have been identified. These subunits contain four transmembrane regions with intracellular NH₂- and COOH-termini. The first extracellular loop contains the conserved region of the GLWXXC amino acid motif, the most distinct feature of all the γ subunits (Catterall, 2011). The γ subunits also regulate biophysical and trafficking properties of the α_1c (Arikkath & Campbell, 2003). However, little is known about the function of the γ subunit on the regulation of LTCCs in VSM.

LTCCs are major targets of second messenger/kinase signaling cascades such as protein kinase A (PKA) and protein kinase C (PKC). Modulation of LTCC activity by these kinases ultimately contributes to control VSM function and vascular reactivity. The modulation of vascular LTCC activity by PKA is controversial. Activation of PKA in VSM has been reported to inhibit, potentiate, or has no effect on LTCC activity (see review by Keef, Hume, & Zhong, 2001). This contrasts with abundant and consistent data, indicating that agonists that stimulate PKA activity typically trigger vasodilation. Surprisingly, it was recently reported that an elevation in extra cellular D-glucose from 5 to 15–20 mM, which is similar to the glucose concentration typically observed in animal models of diabetes and human diabetic patients, potentiates LTCC activity in cerebral VSM via a mechanism that requires PKA (Navedo, Takeda, Nieves-Cintron, Molkentin, & Santana, 2010; Nystoriak et al., 2014). This increase in vascular LTCC activity was correlated with enhanced myogenic tone in response to elevated glucose, thus providing the first example, to our knowledge, of a PKA-mediated vasoconstriction. On the other hand, activation of PKC by phorbol esters and vasoconstrictors acting through G_q-coupled receptors (e.g., ANG II, endothelin-1) results in potentiation of vascular LTCC activity and vasoconstriction (Keef et al., 2001; Weiss & Dascal, 2015). Consistent with this, genetic ablation of PKC protected against ANG II-induced potentiation of LTCC activity and the development of hypertension (Nieves-Cintron, Amberg, Navedo, Molkentin, & Santana, 2008). These genetic studies found that PKC activity was also required for basal and persistent LTCC activity in some VSM (see later and Santana et al., 2008). An integrated view of the specific upstream pathways contributing to PKA- and PKC-mediated LTCC regulation remains to be fully elucidated. Further, work in this area may help to identify novel therapeutic targets to treat pathological conditions associated with LTCC dysfunction such as hypertension (Nieves-Cintron et al., 2008).

Studies combining classical electrophysiology with high-resolution total internal reflection fluorescence microscopy have provided important information regarding the spatial organization of functional LTCCs and resultant Ca²⁺ signal in VSM (Nystoriak, Nieves-Cintron, & Navedo, 2013). Using this approach, elementary Ca²⁺ influx events via LTCCs (i.e., LTCC sparklets) were imaged in VSM, and channel activity was found to occur through distinct loci of low and high activity (Navedo, Amberg, Votaw, & Santana, 2005). The molecular, biophysical, and regulatory properties as well as the functional role of LTCC sparklets in VSM have been extensively reviewed in recent papers (Navedo & Amberg, 2013; Navedo & Santana, 2013; Santana & Navedo, 2009).

LTCC sparklets are sensitive to dihydropyridines and extracellular Ca²⁺ concentration and insensitive to store depletion by thapsigargin. Noteworthy, LTCC sparklets are always associated with inward L-type Ca²⁺ currents, confirming that they are produced by Ca²⁺ influx via LTCC. Whereas low activity LTCC sparklets exhibit stochastic behavior, high activity LTCC sparklets are produced by prolonged channel openings and in many cases by the nonstochastic, coordinated opening of clustered LTCC channels (Navedo, Cheng, et al., 2010). The dual optical/electrical recording of Ca + influx via LTCC also provides critical information regarding functional regulation of these channels. Importantly, the structurally diverse scaffolding protein AKAP150 was shown to be critical in mediating LTCC regulation. By virtue of its ability to bind PKA, PKC, calcineurin, and the LTCC itself, AKAP150 facilitates LTCC regulation by these kinases and phosphatase (Navedo, Amberg, Nieves, Molkentin, & Santana, 2006; Navedo et al., 2008; Navedo & Santana, 2013; Navedo, Takeda, et al., 2010; Santana & Navedo, 2009). High activity LTCC sparklets were shown to require distinct PKC and calcineurin activity. Accordingly, high activity LTCC sparklets contribute to [Ca²⁺]_i, and myogenic tone during physiological and pathological conditions (Amberg, Navedo, Nieves-Cintrón, Molkentin, & Santana, 2007; Takeda, Nystoriak, Nieves-Cintron, Santana, & Navedo, 2011). Indeed, exacerbated high LTCC sparklet activity that is dependent on PKC or PKA has been linked to increased myogenic tone and activation of prohypertensive signaling pathways in animal models of hypertension and diabetes, respectively (Navedo & Amberg, 2013; Navedo, Takeda, et al., 2010; Nieves-Cintron et al., 2008, 2015; Nystoriak et al., 2014).

In summary, splice variations for the α_1c, β, and α₂δ subunits result in complex functional diversity of LTCCs. As the predominant Ca²⁺ entry pathway in VSM, LTCCs play a key role in modulating VSM contractility and myogenic tone. Thus, mechanisms regulating LTCC subunit composition, posttranslational modifications, and membrane organization have the potential to impact VSM function and vascular reactivity during physiological and pathological conditions.

2.1.2 T-Type Ca²+Channels

TTCCs were first identified as a separate VDCC in guinea pig ventricular myocytes as transient conductance currents of ~8pS with Ba²⁺ as the charge carrier (Catterall, 2011). T-type currents are activated at more hyperpolarized potentials (~−30 mV). These channels can be blocked by mibefradil, NNC 55-0396, pimozide, penfluridol, and nickel, although caution should be taken as many of these compounds have off-target effects (Gray & Macdonald, 2006). Dihydropyridines such as nifedipine (at nanomolar range) have generally minimal effects on TTCCs. However, it has been shown that nifedipine at micromolar concentrations (>1 μM) can suppress TTCC function (Akaike et al., 1989). The T-type conductance is similar with Ba²⁺ and Ca²⁺ as charge carriers, whereas L-type current is significantly larger in the presence of Ba²⁺ than with Ca²⁺ (Catterall, 2011). So far, no auxiliary β, α₂δ,or γ subunits have been purified for the TTCC. However, some studies suggest that LTCC auxiliary subunits may modulate TTCC functions (Perez-Reyes, 2006).

Several studies have now suggested a role for TTCCs in VSM physiology and vascular reactivity. At the molecular level, transcript and protein for Ca_V3.1 and Ca_V3.2 have been found in VSM from several vascular beds and in different species, including humans (Abd El-Rahman et al., 2013; Harraz, Abd El-Rahman, et al., 2014; Harraz, Visser, et al., 2015; Kuo, Ellis, Seymour, Sandow, & Hill, 2010). Studies on rat mesenteric arterioles indicate that TTCCs also contribute to vasoconstrictor responses (Gustafsson, Andreasen, Salomonsson, Jensen, & Holstein-Rathlou, 2001; Jensen, Salomonsson, Jensen, & Holstein-Rathlou, 2004). In skeletal muscle arteries, Ca_V3.1 and Ca_V3.2 are actively involved in maintenance of myogenic tone (VanBavel, Sorop, Andreasen, Pfaffendorf, & Jensen, 2002). In arteriolar SM from the retina, Ca_V3.1 activity has been reported, indicating a potentially important role in retinal microcirculation (Fernandez, McGahon, McGeown, & Curtis, 2015).

Ca_V3.1 and Ca_V3.2 channels have also been identified in rat and mouse cerebral VSM as the nifedipine-insensitive component of Ba²⁺ currents (Abd El-Rahman et al., 2013; Harraz, Abd El-Rahman, et al., 2014; Harraz, Visser, et al., 2015; Kuo et al., 2010; Nikitina et al., 2006). Interestingly, Ca_V3.1 seems to be replaced by Ca_V3.3 in human cells (Harraz, Visser, et al., 2015). Ca_V3.1/Ca_V3.3 and Ca_V3.2 contributions to the observed T-type current in these cells could be distinguished based on a 20-fold higher sensitivity of Ca_V3.2 to blockage by Ni²⁺ (Ca_V3.2 EC₅₀ = 12 μM; Ca_V3.1 EC₅₀ = 250 μM) (Lee, Gomora, Cribbs, & Perez-Reyes, 1999). By exploiting this selectivity and the use of genetically modified mice, the contribution of these channels to the regulation of the myogenic response was found to diverge (Fig. 1). Whereas Ca_V3.1/Ca_V3.3 seems to mediate pressure-induced constriction, Ca_V3.2 contributes to negative feedback regulation of pressure-induced tone by modulating the RyR–large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel axis (Harraz, Abd El-Rahman, et al., 2014; Harraz, Brett, & Welsh, 2014; Harraz, Visser, et al., 2015). Another interesting detail is that TTCCs and LTCCs appear to respond to different intravascular pressures following their voltage dependence (Harraz, Abd El-Rahman, et al., 2014; Harraz, Visser, et al., 2015). Accordingly, Ca_V3.1/Ca_V3.3 channels predominantly contribute to myogenic tone at lower intraluminal pressures (e.g., 20–40 mmHg), in which membrane potential of VSM is ~ −60 to −50 mV. Conversely, Ca_V1.2 function is prominent at more depolarized VSM membrane potentials (~−45 to −36 mV) observed at greater intraluminal pressures (Knot & Nelson, 1998). Regulation of TTCC activity by protein kinases may also contribute to modulate VSM function and the myogenic response. Accordingly, PKA and PKG activation has been shown to inhibit TTCC in VSM (Harraz, Brett, et al., 2014; Harraz & Welsh, 2013a). This TTCC suppression could limit extracellular Ca²⁺ entry, which may contribute to the well-known vasodilatory responses triggered by these kinases. Thus, along with LTCCs, TTCCs may contribute to precise maintenance of myogenic tone through their ability to activate at lower pressures.

2.2 TRP Channels

TRP channels are a superfamily of cationic channels with 28 encoding genes. Based on their sequence homology, these channels can be further categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPA (ankyrin), and TRPML (mucolipin) (Earley & Brayden, 2015). Sequence analysis suggests that TRP channels consist of six membrane-spanning helices (S1–S6) with intra-cellular NH₂- and COOH-termini of variable lengths. While a crystal structure has not yet been resolved for any TRP channel, electron cryomicroscopy studies of the capsaicin-activated TRPV1 channel demonstrate four symmetrical subunits with S5 and S6 loop forming the ion pore. The structure also contains a selectivity filter, which is differentially regulated by endogenous and exogenous ligands (Liao, Cao, Julius, & Cheng, 2013). Functional TRP channels consist of four subunits and can be homomeric or heteromeric in nature. As most cells express multiple TRP channel isoforms, it is likely that these channels exist in heteromultimeric form. The NH₂- and COOH-termini have several domains that can modify and regulate channel function. For example, the number of ankyrin repeats on the TRPV1 and TRPA1 channels has been shown to regulate channel activity (Gaudet, 2008). Similarly, the COOH-terminus of some TRP channels contains a CaM/IP₃-binding domain, serine–threonine kinase target sequence, and PDZ protein–protein interaction domains, depending on transcript splicing patterns (Walker, Hume, & Horowitz, 2001).

Multiple TRP channels are expressed in VSM. In these cells, TRP channels contribute to regulation of membrane potential, contraction, and development of myogenic tone (Earley & Brayden, 2015). Additionally, certain TRP channels contribute to vascular mechanosensitivity via G-protein-coupled signaling in resistance arteries (Earley & Brayden, 2015). Almost all TRP channels are permeable to Ca²⁺ with the exception of TRPM4 and TRPM5, which are Ca²⁺ activated, but not Ca²⁺ permeable (Earley & Brayden, 2015). Here, we review key issues on specific vascular TRP channels.

2.2.1 TRPV1

TRPV1 are nonselective cation channels with the preference for Ca²⁺ to Na⁺ ions (10:1) (Caterina et al., 1997). TRPV1 channels are primarily expressed in sensory neurons and play a crucial role in heat sensation and nociception (Meents, Neeb, & Reuter, 2010). TRPV1 can be endogenously activated by acidic pH < 5.5 and derivatives of arachidonic acid, and exogenously by capsaicin and resiniferatoxin. Furthermore, channel activity and trafficking are regulated by PKA and PKC (Efendiev, Bavencoffe, Hu, Zhu, & Dessauer, 2013; Koda et al., 2016) with potential important implications in the regulation of vascular tone (Earley & Brayden, 2015). Activation of these channels in sensory nerves results in release of vasodilatory neuropeptides and consequently, dilation of the blood vessels. In SM, TRPV1 activation elicits contraction. For example, skeletal muscle arterioles exhibit significant constriction in response to application of the TRPV1 activator capsaicin (Toth et al., 2014). TRPV1 expression has also been reported in arterioles from thermoregulatory tissues including dura, cremaster, skin, and ear (Cavanaugh et al., 2011). Yet, TRPV1 expression does not seem to be ubiquitous among vascular tissues (Baylie & Brayden, 2011).

2.2.2 TRPV2

TRPV2 channels have been reported in aortic, mesenteric, and basilar artery VSM (Earley & Brayden, 2015). In aortic myocytes, application of a hypotonic solution resulted in TRPV2 activation, increased Ca²⁺ influx, and constriction (Muraki et al., 2003). Yet, more studies are required to elucidate a functional role for TRPV2 in different vascular beds.

2.2.3 TRPV4

TRPV4 channels have been implicated in regulation of myogenic tone. This channel can be stimulated by mechanical stress, including sheer stress and cell swelling. In cerebral VSM, TRPV4 channels are regulated by PKC and their activation results in Ca²⁺ influx (i.e., TRPV4 sparklets) (Mercado et al., 2014). Unexpectedly, this activation was associated with vasodilation rather than vasoconstriction (Earley, Heppner, Nelson, & Brayden, 2005). Ca²⁺ entry during a single TRPV4 sparklet is ~ 100-fold larger than a Ca_V1.2 sparklet (Mercado et al., 2014). This highly localized TRPV4 sparklet may stimulate the activity of RyR in the SR resulting in generation of Ca²⁺ sparks. The ensuing Ca²⁺ sparks activate BK_Ca channels leading to VSM membrane potential hyperpolarization and vasorelaxation (Fig. 1) (Earley et al., 2005, 2009). An intriguing possibility, based on the large and localized flux of Ca²⁺ through TRPV4, is that these channels could also directly activate nearby BK_Ca channels to regulate vascular reactivity. This hypothesis requires examination.

2.2.4 TRPC1

TRPC1 channels form functional heteromers with TRPC4 and/or TRPC5, which can be regulated by G_q-coupled signaling pathways (Sabourin et al., 2009; Strubing, Krapivinsky, Krapivinsky, & Clapham, 2001). The functional relevance of TRPC1 channels in VSM, however, is controversial. For instance, studies support (Bergdahl et al., 2003, 2005; Inoue et al., 2006)and refute (DeHaven et al., 2009; Dietrich et al., 2007; Varga-Szabo et al., 2008) a role for TRPC1 in store-operated Ca²⁺ entry (SOCE) in VSM. Moreover, Ca²⁺ influx via TRPC1 has been associated with BK_Ca channel activation and VSM relaxation (Kwan et al., 2009). This is somewhat paradoxical as a recent study suggested that (1) TRPC1 does not form functional homomeric channels and (2) TRPC1-containing heteromers exhibit decreased Ca²⁺ permeability (Storch et al., 2012). More research is necessary to define the mechanisms by which TRPC1 modulates BK_Ca channel activity and vascular reactivity.

2.2.5 TRPC3

TRPC3 in VSM has been linked to vascular tone regulation via stimulation of a variety of G-protein-coupled receptors (GPCRs) including ANG II and endothelin-1 (ET-1) receptors (Earley & Brayden, 2015). Accordingly, TRPC3 does not seem to have an effect on intrinsic vascular tone, suggesting that TRPC3 may not be essential in pressure-induced tone but rather receptor-mediated vasoconstriction of the arteries (Reading, Earley, Waldron, Welsh, & Brayden, 2005; Xi et al., 2008). Indeed, in cerebral VSM, the mechanism of TRPC3-induced vasoconstriction implicates IP₃ facilitated coupling of IP₃R and TRPC3 (Fig. 1)(Xi et al., 2008). The activation of TRPC3 results in cation influx (e.g., Na⁺ and Ca²⁺) and consequently membrane depolarization leading to opening of LTCCs and vasoconstriction.

2.2.6 TRPC4

A role for TRPC4 in aortic and mesenteric VSM has been suggested (Lindsey & Songu-Mize, 2010). Under prolonged cyclic stretch conditions, TRPC4 expression appears to decrease along with a decrease in SOCE. These studies suggest that downregulation of TRPC4 may be a protective mechanism against stretch-mediated increase in SOCE.

2.2.7 TRPC5

TRPC5 channels have been implicated in SOCE in VSM when coassembled with other TRPC subunits. For example, application of an anti-TRPC5 antibody was found to inhibit SOCE in VSM from cerebral arterioles in response to store depletion (Xu, Boulay, Flemming, & Beech, 2006). Likewise, currents evoked by cyclopiazonic acid (a SERCA pump inhibitor) were inhibited by an anti-TRPC5 antibody (Saleh, Albert, & Large, 2009). Altogether, these results suggest a role for TRPC5 in SOCE in VSM.

2.2.8 TRPC6

TRPC6-mediated Ca²⁺ mobilization in VSM has been associated with regulation of vasoconstriction. TRPC6 channels can be activated via mechanosensation, such as cell swelling and sheer stress, to promote vasoconstriction (Welsh, Morielli, Nelson, & Brayden, 2002). These channels can also be activated by GPCRs (Inoue et al., 2001). For example, application of 1 nM ANG II activates TRPC6 channels via a mechanism that is directly associated with diacylglycerol, and independent of PKC (Helliwell & Large, 1997; Saleh, Albert, Peppiatt, & Large, 2006). More recently, an exciting study proposed that Ca²⁺ entry via TRPC6 plays a critical role as part of a force-sensing complex that bolsters Ca²⁺ release through IP₃Rs on the SR to stimulate TRPM4 channel activity and myogenic tone (Fig. 1)(Gonzales et al., 2014). However, despite the wealth of information on this channel in VSM, additional studies are needed to establish their (1) contributions to local and global Ca²⁺ signals, (2) role in different vascular beds, and (3) mechanisms of activation and mechanosensation.

2.2.9 TRPM4

TRPM4 is one of two TRP channels that are Ca²⁺ activated, but not Ca²⁺ permeable. Nonetheless, TRPM4 channels in VSM play an essential role in development of myogenic tone (Earley, Waldron, & Brayden, 2004; Gonzales et al., 2014; Li & Brayden, 2015). In VSM from rat cerebral arteries, these channels are activated by local IP3R-mediated increases in [Ca²⁺]_i (Fig. 1)(Gonzales, Amberg, & Earley, 2010). Their activity can be differentially regulated by specific signaling proteins depending on the vascular bed (Earley, Straub, & Brayden, 2007; Li & Brayden, 2015). More recently, RhoA/Rho-associated protein kinase has been demonstrated to potentiate TRPM4 in VSM of parenchymal arterioles (Li & Brayden, 2015). Other TRPM channels, such as TRPM7 and TRPM8, are also expressed in VSM. Whereas TRPM7 has been implicated mainly in Mg²⁺ homeostasis (He, Yao, Savoia, & Touyz, 2005), little is known about the functional role of TRPM8 in VSM.

2.2.10 TRPP2

TRPP are Ca²⁺-permeable channels known to be mechanosensitive (Earley & Brayden, 2015). Recognized also as polycystic-1 and -2 proteins, TRPP1 and TRPP2 expression has been described in multiple vascular beds, including mesenteric and cerebral arteries (Narayanan et al., 2013; Sharif-Naeini et al., 2009). In these arterial beds, the activation of TRPP1 and TRPP2 seems to regulate the myogenic response, albeit via different mechanisms. TRPP2 activation is also associated with differential regulation of IP₃R and RyR in cells (presumably VSM) from cerebral arteries with important implications for modulation of vascular reactivity (Abdi et al., 2015). Yet, several issues still require further examination such as (1) contribution of TRPP2 to local and global Ca²⁺ signals, (2) how TRPP2 modulates IP₃R and RyR activity, (3) what role, if any, does interaction of TRPP2 with other ion channels (e.g., TRPP1) have on the control of vascular reactivity, and (4) how TRPP2 may contribute to VSM physiology and pathophysiology.

2.2.11 Intracellular TRP Channels

Accumulating evidence suggests that TRP channels in intracellular membranes may also play a critical role in regulating Ca²⁺ homeostasis and cell function (Dong, Wang, & Xu, 2010). For example, TRPV1 and TRPP2 localized to the endoplasmic reticulum are thought to be involved in intracellular Ca²⁺ regulation (Koulen et al., 2002; Olah et al., 2001). Furthermore, TRPM2 and TRPML may play a role in Ca²⁺ release from lysosomes, which may contribute to oxidative stress of the cell (Dong et al., 2010; Lange et al., 2009). In VSM, TRPM7-containing vesicles are quickly trafficked to the plasmalemma in response to shear stress (Oancea, Wolfe, & Clapham, 2006). This resulted in a significant increase in TRPM7-like currents, which may contribute to increased [Ca²⁺]_i and impaired VSM function during pathological conditions (Dong et al., 2010). Extensive studies, however, are still required to completely understand the role of intracellular TRP channels in VSM.

2.3 Orai and STIM

Depletion of Ca²⁺ from intracellular stores via activation of plasmalemmal phospholipase C (PLC)-coupled receptors and subsequent IP₃R-mediated Ca²⁺ release triggers Ca²⁺ influx from extracellular sources. This process of SOCE was first introduced as a mechanism of controlled and sustained Ca²⁺ entry following activation of surface membrane receptors (Putney, 1986). The physiological and pathological significance of the Ca²⁺ release-activated Ca²⁺ current (I_CRAC) was brought to light by rare cases of severe immunodeficiency in patients with inherited defects in components mediating SOCE that profoundly impairs immune cell function (Picard et al., 2009). A great deal of progress has been made in the field of SOCE and has highlighted an emerging importance of I_CRAC in multiple cell types, including VSM. Following decades-long investigations aimed at revealing the molecular identity of I_CRAC, the plasmalemmal Ca²⁺-permeable channels and associated S/ER-localized channel activators responsible for SOCE were only recently identified as Orai and STIM, respectively (Roos et al., 2005; Zhang et al., 2005). It is now recognized that upon reduction of Ca²⁺ concentration in S/ER ([Ca²⁺]_S/ER), the [Ca²⁺]_S/ER sensor STIM1 undergoes dynamic spatial reorganization into aggregate clusters that interact with Orail channels in the plasmalemma to facilitate I_CRAC. This section will briefly review the current state of knowledge regarding molecular and functional aspects of Orai-channel mediated Ca²⁺ signaling in VSM and its potential contribution to vascular disease states.

The Orai homologues are plasmalemmal ion channels encoded by three genes (i.e., Orai1, Orai2, and Orai3) with little genetic or structural similarity to that of other known Ca²⁺-permeable channels. In mammals, alternative methionine translation initiation gives rise to two forms of Orai1: a 33 kDa long form (Oria1α) and a 23 kDa short form (Oria1β) (Fukushima, Tomita, Janoshazi, & Putney, 2012). Perhaps as a result of these proteomic and functional differences, Orai1α and Orai1 β could display preference for certain binding partners (e.g., TRPC1 vs Orai3) to exhibit selective participation in distinct Ca²⁺ currents (Desai et al., 2015). Sequence analyses and crystallization studies have revealed that Orai channels are heteromeric structures, with each channel consisting of 4–6 Orai subunits (Hou, Pedi, Diver, & Long, 2012). Each subunit consists of four highly conserved transmembrane helices (M1–M4). The side chains of amino acids in M1 helices of each Orai subunit form a 55 A pore. The extracellular face of the Orai pore has a distinct ring of glutamate residues that form its selectivity filter (Hou et al., 2012). This feature is thought to render the highly selective nature of I_CRAC being almost exclusively carried by Ca²⁺ over Na⁺ or K⁺ ions (Hoth & Penner, 1993). Each Orai subunit consists of cytosolic NH₂ and COOH-termini, which contain sites for functional regulation by Ca²⁺/CaM, PKC, and STIM proteins (Frischauf et al., 2009; Hooper et al., 2015; Mullins, Park, Dolmetsch, & Lewis, 2009).

It is now established that Orai channels are activated via physical molecular interaction with the stromal interaction molecules (STIM1 and STIM2), which function as Ca²⁺ sensors within the S/ER (Roos et al., 2005; Zhang et al., 2005). STIM proteins are single-transmembrane proteins that are primarily located in the ER, although small populations of STIM1 that play a role in controlling Ca²⁺ entry have also been observed in the plasmalemma (Spassova et al., 2006). S/ER STIM senses alterations in luminal [Ca²⁺]_i via NH₂ terminal canonical EF-hand domains. Upon a depletion of S/ER Ca²⁺ and dissociation of Ca²⁺ ions from the NH₂ terminal EF-hand domains, STIM proteins undergo unfolding and aggregation at plasmalemmal-S/ER junctions where they activate Orai channels via direct physical interactions to induce conformational changes in the channel structure. Importantly, proper Orai/STIM communication requires COOH-terminus coiled-coil interaction domains of both Orai and STIM (Frischauf et al., 2009; Muik et al., 2008).

Determining the precise physiological role of the SOCE machinery in cardiovascular tissues has been hampered by a lack of selective pharmacological modulators of Orai channels and STIM proteins. The widely used nonselective cation channel inhibitor 2-aminoethoxydiphenyl borate (2-APB) has been shown to exhibit concentration-dependent and divergent effects on I_CRAC (Prakriya & Lewis, 2001). A new class of I_CRAC inhibitors was shown to have selective effects on Orai channels independent of STIM oligomerization or STIM/Orai interaction (Derler et al., 2013). For example, in VSM, the I_CRAC inhibitor S66 prevented Ca²⁺ influx following store depletion with nanomolar potency (Li et al., 2011). Thus, the emergence of novel compounds that can specifically target Orai–Orai/STIM interactions and STIM aggregation will significantly aid in future studies to investigate I_CRAC-related mechanisms in cardiovascular physiology and pathology.

Expression of Orai and STIM ranges from very low to nondetectable in quiescent VSM. Yet, store depletion by thapsigargin and subsequent SOCE was shown to be prominent in synthetic cultured rat aortic smooth muscle, but not in freshly dispersed VSM (Potier et al., 2009). In line with enhanced SOCE in dedifferentiated SM present in many disease states, transformation of contractile VSM to a noncontractile proliferative phenotype in culture is associated with substantial upregulation in the expression of Orai and STIM proteins (Berra-Romani, Mazzocco-Spezzia, Pulina, & Golovina, 2008; Potier et al., 2009). Consistent with in vitro findings, expression for both Orai1 and STIM1 were strongly upregulated in association with SM proliferation following balloon angioplasty-induced carotid injury in rats (Zhang et al., 2011). Lentivirus-mediated knockdown of Orai1 in injured vessels prevented conversion of SM to a proliferative phenotype and mitigated neointima formation, suggesting that Orai1-mediated Ca²⁺ entry may be an important determinant of vascular remodeling during injury such as in restenosis. Further, in vitro studies have confirmed that SOCE becomes a predominant source of Ca²⁺ influx in synthetic VSM that plays a pivotal role in proliferative and migratory processes. Thus, adaptive changes in Orai and STIM expression and function may drive phenotypic modulation during angiogenesis and vascular repair, as well as in disease.

The precise molecular determinants underlying Orai/STIM up-regulation in phenotypic switching of quiescent to proliferative/migratory SM is still unresolved. A major driving factor of phenotypic switching of VSM is the polypeptide platelet-derived growth factor (PDGF). PDGF, via activation of the PDGFβ receptor and downstream PLCγ-mediated SR Ca²⁺ release, is an important activator of Orai1 channels in SM. Whether STIM-independent regulation of Orai occurring downstream of PDGF stimulation significantly contributes to Ca²⁺ influx in proliferating/migratory VSM is still unclear. However, in addition to the well-known role in activation of Orai channels, STIM1 clustered in ER/PM junctions also inhibits Ca²⁺ influx through Ca_V1.2 channels and leads to internalization of LTCC (Park, Shcheglovitov, & Dolmetsch, 2010; Wang et al., 2010). This mechanism, however, could not be confirmed in quiescent VSM (Takeda et al., 2011), perhaps reflecting distinct roles for STIM proteins in proliferative vs contractile cells.

It is also important to mention the role that Orai channels play in store-independent, ligand-activated Ca²⁺ entry in SM that is mediated by Orai1, Orai3, and STIM1. This nonstore-operated strongly inwardly rectifying current, termed I_ARC, is gated by arachidonic acid and its metabolite leukotriene C4 (Zhang et al., 2015). In VSM, Ca²⁺ entry requiring Orai1, Orai3, and STIM1 has been observed independent of sustained store depletion following application of the proinflammatory peptide thrombin. Like Orai1 and STIM1, Orai3 protein was also upregulated in an experimental model of carotid artery injury and in vivo knockdown of this subunit alone, blunted neointima formation. These findings suggest that heteromultimerization of Orai channels could give rise to store-dependent and store-independent Ca²⁺ entry pathways that could contribute to maintenance of the synthetic VSM phenotype in several disease states.

3. SR Ca²⁺ CHANNELS

Ca²⁺ release channels located on the SR membrane of VSM play pivotal roles in controlling cell excitability and vascular reactivity. The two major classes of Ca²⁺ release channels in VSM are RyR and IP₃R. In the following section, we describe their role in VSM.

3.1 Ryanodine Receptors

RyRs are intracellular Ca²⁺ channels that mediate Ca²⁺ release from the SR. Three RyR isoforms (RyR1–RyR3) encoded by three distinct genes have been identified (Lanner, Georgiou, Joshi, & Hamilton, 2010). Structural models predicted both NH₂- and COOH-termini to be cytosolic and the pore to have 4–12 membrane-spanning domains (Lanner et al., 2010). Single-particle electron cryomicroscopy studies have provided insights into channel gating and interaction with modulators (Samso, Wagenknecht, & Allen, 2005; Serysheva et al., 2008). Recently, 4.8 and 6.8 A resolution structures of the RyR were described by two independent groups (Efremov, Leitner, Aebersold, & Raunser, 2015; Zalk et al., 2015). Both reports suggest that Ca²⁺ sensitivity of RyR1 is imparted by EF-hand domains in α-solenoid structures that connect the cytoplasmic region to the channel pore.

mRNA transcript and protein levels have been detected for all RyR isoforms in VSM. RyR1 and RyR2 mediate Ca²⁺ sparks (e.g., Ca²⁺ release from intracellular stores through RyRs) in portal vein SM (Coussin, Macrez, Morel, & Mironneau, 2000), whereas RyR2 has been found to be the predominant isoform in VSM from rat resistance vessels (Vaithianathan et al., 2010). Several studies suggest that in contrast to the tight coupling between LTCCs and RyRs in skeletal and cardiac muscle, a loose coupling mechanism may exist in VSM in which LTCCs indirectly modulate RyRs by contributing to global [Ca²⁺]_i and SR Ca²⁺ load (Collier, Ji, Wang, & Kotlikoff, 2000; Essin et al., 2007). Interestingly, recent studies demonstrate that application of Ni²⁺ at a concentration that selectively inhibits Ca_V3.2 reduced Ca²⁺ spark activity in VSM from WT mice, but had no effect in cells from Ca_V3.2 knockout mice (Harraz, Brett, et al., 2015). Consistent with a role for Ca_V3.2 in modulation of Ca²⁺ sparks, these channels were found juxtaposed with RyR in specific microdomains (Harraz, Abd El-Rahman, et al., 2014), suggesting that Ca²⁺ influx through TTCCs may contribute to RyR activation in VSM (Fig. 1). This may represent a novel mechanism for regulation of RyRs, VSM excitability, and vascular reactivity that requires further examination.

In VSM, RyR can be activated by caffeine, and depending on its concentration, it can induce massive Ca²⁺ release from intracellular stores or increase the frequency of Ca²⁺ sparks (Jaggar, Porter, Lederer, & Nelson, 2000). The receptor can be inhibited in a concentration-dependent manner by the alkaloid ryanodine. Accordingly, at low concentrations, ryanodine can activate RyR, while at higher concentrations, it inhibits the receptor. Other pharmacological agents such as tetracaine have been used to block RyR and examine their role in VSM function. However, their use is limited due to nonspecific effects. RyRs play a central role in excitation–contraction coupling in both skeletal and cardiac muscle where they contribute to the global increase in [Ca²⁺]_i necessary for contraction. However, RyRs influence VSM excitability indirectly by modulating the activity of plasmalemma ion channels (Fig. 1). In a landmark study, it was found that Ca²⁺ sparks could simultaneously activate multiple BK_Ca channels in the plasmalemma to produce spontaneous transient outward currents (STOCs) and promote hyperpolarization and relaxation of VSM in small resistance arteries (Nelson et al., 1995; Perez, Bonev, Patlak, & Nelson, 1999). The spatial proximity between the plasmalemma and the SR in VSM (Somlyo, 1985) permits Ca²⁺sparks to activate BK_Ca channels with minimal effects on global [Ca²⁺]_i. In rabbit portal vein, however, RyRs have been found to depolarize VSM through activation of Ca²⁺ sensitive chloride channels (Saleh & Greenwood, 2005; Wang, Hogg, & Large, 1992). This highlights the importance of RyR in fine-tuning VSM excitability among different vascular beds.

Regulation of the functional coupling between RyR and BK_Ca channels has profound implications for VSM function. For instance, the vasodilatory effects of nitric oxide and forskolin can be attributed, at least in part, to an increase in PKG and PKA activity that acts on RyR to stimulate Ca²⁺ sparks-mediated STOC frequency (Jaggar et al., 2000). Conversely, activators of PKC inhibit RyR activity, which reduces STOC frequency and could contribute to vasoconstriction (Amberg et al., 2007; Bonev, Jaggar, Rubart, & Nelson, 1997). In addition, any disturbance on BK_Ca channel Ca²⁺ sensitivity may impact functional RyR–BK_Ca coupling, and vascular contractility; a point well illustrated in animal models of hypertension. In these animals, decreased expression of BK_Ca channel β1 subunit reduces BK_Ca Ca²⁺ sensitivity resulting in impaired STOC activity, increased myogenic tone, and hypertension (Amberg, Bonev, Rossow, Nelson, & Santana, 2003; Nieves-Cintron, Amberg, Nichols, Molkentin, & Santana, 2007). The coupling strength between RyR and BKCa channel is also affected in animal models of diabetes (Nystoriak et al., 2014). These examples highlight the relevance of the relationship between RyR and BK_Ca channels in VSM with impaired communication leading to vascular dysfunction.

3.2 Inositol-1,4,5,-Trisphosphate Receptors

IP₃R is a ubiquitously expressed Ca²⁺ release channel localized to the SR membrane (Narayanan et al., 2012; Nixon, Mignery, & Somlyo, 1994). These channels consist of four membrane-spanning subunits surrounding the central ion permeation pore. Each subunit contains six transmembrane domains, a luminal loop that forms the ion-conducting pore between transmembrane domains 5 and 6, and cytosolic NH₂- and COOH-termini. The NH₂-terminus is further subdivided into a suppression domain that inhibits IP₃ binding, an IP₃-binding core domain, binding sites for ATP and Ca²⁺, and a coupling domain for physical interactions with TRPC channels. The COOH-terminus seems to contribute to IP₃R tetramerization, and recent electron cryomicroscopy studies with a resolved IP₃R structure at 4.7 Å implicated this domain in channel gating (Fan et al., 2015). Moreover, these studies also suggest that the gate for the Ca²⁺ conduction path includes several hydrophobic residues located closer to the cytosolic side of the SR membrane (Fan et al., 2015). Yet, further experiments will be needed to completely understand the permeation, gating, and regulatory mechanisms governing IP₃R function.

Three different isoforms of IP₃R (IP₃R1, IP₃R2, IP₃R3) have been reported (Narayanan et al., 2012). While expression of all three isoforms has been found in VSM from aorta, mesenteric, and cerebral arteries, IP₃R1 seems to be the predominant isoform in VSM from small resistance arteries (Grayson, Haddock, Murray, Wojcikiewicz, & Hill, 2004; Zhao, Adebiyi, Blaskova, Xi, & Jaggar, 2008; Zhou et al., 2008). Yet, the expression profile of the IP₃R isoforms can vary depending on the developmental stage. Accordingly, expression of IP₃R3 is high in neonatal SM, decreases during development, and is surpassed by increasing IP₃R1 expression in adult SM (Tasker, Michelangeli, & Nixon, 1999). High levels of IP₃R2 and IP₃R3 expression have also been found in proliferating SM (Tasker, Taylor, & Nixon, 2000). These IP₃R isoforms also differ in IP₃-binding affinities as follows: IP₃R2 > IP₃R1 > IP₃R3 (Newton, Mignery, & Sudhof, 1994; Wojcikiewicz & Luo, 1998). Physical localization of IP₃R on the SR membrane is also isoform and tissue-dependent, which may be important for distinct physiological functions such as gene expression and VSM excitability (Nixon et al., 1994; Tasker et al., 2000; Zhao et al., 2008).

IP₃R activation is stimulated by the second messenger IP₃, which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC in response to activation of Gq/11-coupled receptors. Indeed, IP3R activity in VSM can be stimulated by many endogenous vasoactive molecules that act through Gq/11-coupled receptors to produce IP3, including ET-1, acetylcholine, noradrenaline, and serotonin (Berridge, 2008). Pharmacological inhibition of IP₃R can be achieved with the application of widely used agents such as 2-APB and xestospongin C. IP₃R inhibition may have distinct effects on vascular reactivity. For instance, in mouse mesenteric arteries, IP₃R inhibition with xestospongin C did not affect the myogenic tone, but did prevent the phenylephrine-induced vasoconstriction (Mauban, Zacharia, Fairfax, & Wier, 2015). IP₃R activity in VSM can be modulated by [Ca²⁺]_i, luminal SR Ca²⁺ load, ATP, several protein kinases (e.g., PKA, PKG), regulatory proteins (e.g., RACK, FKBP12), reactive oxygen species (ROS), and pH (Bezprozvanny, Watras, & Ehrlich, 1991; Iino, 1990; Narayanan et al., 2012).

The activation of IP₃R can produce multiple Ca²⁺ signals, including Ca²⁺ puffs and Ca²⁺ waves, with important implications for VSM function. Ca²⁺ puffs are elementary, localized Ca²⁺ release events produced by clusters of IP₃R(Parker & Smith, 2010; Tovey et al., 2001). Ca²⁺ puffs have been observed in colonic and ureteric SM (Boittin et al., 2000; Olson, Chalmers, & McCarron, 2010), but not in VSM, perhaps due to differences in IP₃R localization, distribution, and function. Yet, recent indirect evidence suggests that Ca²⁺ puffs could alter cerebral VSM function through modulation of plasmalemmal ion channel activity. Accordingly, localized Ca²⁺ release via IP₃R was shown to promote the opening of TRPM4 channels leading to pressure-induced membrane depolarization and cell contraction (Fig. 1)(Gonzales et al., 2010; Gonzales & Earley, 2012).

On the other hand, Ca²⁺ waves are propagating elevations in [Ca²⁺]_i resulting from Ca²⁺ release via IP₃R, RyR, or both due to electrical, mechanical, and receptor-mediated stimulation in VSM (Amberg & Navedo, 2013; Narayanan et al., 2012; Wray & Burdyga, 2010). The contributions of IP₃R and/or RyR to spontaneous and agonist-induced Ca²⁺ wave generation seem to differ in VSM according to the vascular bed (Fig. 1) (Boittin, Macrez, Halet, & Mironneau, 1999; Dabertrand, Nelson, & Brayden, 2012; Gordienko & Bolton, 2002; Jaggar, 2001; Wray & Burdyga, 2010; Zacharia, Zhang, & Wier, 2007; Zhao et al., 2008). For example, RyR but not IP₃R was found to play a prominent role in spontaneous Ca²⁺ wave generation and propagation in cerebral VSM (Jaggar, 2001; Jaggar & Nelson, 2000), whereas both RyR and IP₃R appear to be involved in Ca²⁺ waves in portal vein SM (Gordienko & Bolton, 2002). Conversely, IP₃Rs are involved in agonist-induced Ca²⁺ waves, which may propagate with involvement of RyR activation perhaps via a Ca²⁺-induced Ca²⁺ release (CICR) mechanism (Boittin et al., 1999; Gordienko & Bolton, 2002; Wray & Burdyga, 2010; Zhao et al., 2008). The mechanisms of wave propagation, however, remain unclear. Future studies should comprehensively investigate the levels of RyR and IP₃R expression, subcellular distribution and activation, their regulation by cytosolic and SR Ca²⁺ concentration, as well as variations in these properties in VSM among different vascular beds.

IP₃R-mediated SR Ca²⁺ release-dependent and -independent mechanisms have been described to regulate VSM function. IP₃R-mediated Ca²⁺ waves have been suggested to contribute to agonist-induced VSM contraction and the myogenic response (Boittin et al., 1999; Lamont & Wier, 2004; Zacharia et al., 2007; Zhao et al., 2008). This response seems to involve an increase in the frequency of Ca²⁺ waves that could contribute, at least in part, to elevate global [Ca²⁺]_i to activate the contractile machinery (Hill-Eubanks, Werner, Heppner, & Nelson, 2011). Interestingly, recent studies have also revealed a significant contribution for IP₃R to VSM excitability that is independent of its ability to mediate SR Ca²⁺ release. At physiological intravascular pressures, PLC-coupled receptors promote vasoconstriction by activating a cation current (I_Cat) that requires physical coupling between TRPC3 and IP₃R(Adebiyi et al., 2010; Xi et al., 2008; Zhao et al., 2008).

IP₃R-mediated Ca²⁺ waves have also been proposed to contribute to pressure-induced vasoconstriction, at least at low intravascular pressures, in cerebral VSM (Adebiyi et al., 2010; Gonzales et al., 2014; Mufti et al., 2010; Xi et al., 2008). Two recent studies proposed a role for PLCγ1 in this process, albeit via distinct signaling pathways. One study implicates pressure-induced stimulation of PLCγ1 activity to TRPC6-mediated Ca²⁺ influx leading to activation (via CICR) of IP₃-sensitized IP₃R. The resulting localized rise in [Ca²⁺]_i activates neighboring TRPM4 channels to depolarize VSM and contribute to development of the myogenic response (Gonzales et al., 2014). A second study suggested the involvement of integrin α_νβ₃ in activation PLCγ1, IP₃ production, IP₃R activation, and Ca²⁺ waves generation in response to an increase in intravascular pressure (Mufti et al., 2015). The ensuing Ca²⁺ waves facilitate MLC₂₀ phosphorylation and development of myogenic tone. In principle, these two IP₃R-mediated SR Ca²⁺ release-dependent mechanisms could synergize to contribute to the regulation of pressure-induced vasoconstriction. Future studies should be designed to test this possibility. Additionally, a somewhat counterintuitive role for IP₃,IP₃R, and IP₃R-mediated SR Ca²⁺ release on activation of BK_Ca channels in cerebral VSM has been described. IP₃R activation was found to increase BK_Ca Ca²⁺ sensitivity (Zhao et al., 2010). This was suggested to facilitate BK_Ca channel activity in response to IP₃R-mediated SR Ca²⁺ release to ameliorate agonist-induced vasoconstriction. Thus, multiple IP₃R-mediated SR Ca²⁺ release-dependent and -independent mechanisms can converge to regulate VSM function.

Proliferation of VSM seems to depend on IP₃R-mediated Ca²⁺ release, specifically increased frequency of Ca²⁺ waves (Wilkerson, Heppner, Bonev, & Nelson, 2006). Accordingly, suppression of IP₃R1 expression in A7r5 cell line prevented them from proliferating (Y. Wang et al., 2001). Furthermore, it was found that IP₃R-mediated Ca²⁺ waves are necessary for the dedifferentiation of native VSM from the contractile to proliferative state (Wilkerson et al., 2006), although the mechanisms require further examination.

IP3R-mediated signaling has been proposed to contribute to vascular pathology. For example, mesenteric VSM of ANG II-induced hypertensive mice and spontaneously hypertensive rats display elevated mRNA and protein levels of IP₃R1 (Abou-Saleh et al., 2013). This increased expression was associated with sensitization of IP₃R-mediated Ca²⁺ release, resulting in augmented vasoconstriction in response to stimulation by vasoactive agents in ANG II-induced hypertensive mice. A different study found that increased TRPC3 expression and coupling between TRPC3 and IP3R, but no changes in IP₃R expression, contributed to agonist-induced vasoconstriction during hypertension, and that this did not require IP₃R-mediated SR Ca²⁺ release (Adebiyi et al., 2012). Some of the disparities can be related to the use of different animal models of hypertension, evaluation of different proteins, and/or diverse experimental conditions and approaches. Impaired IP₃R expression, function, and IP₃R-mediated Ca²⁺ signals in VSM have also been documented to contribute to vascular dysfunction during diabetes and atherosclerosis (Massaeli, Austria, & Pierce, 1999; Searls, Loganathan, Smirnova, & Stehno-Bittel, 2010). Thus, IP₃R-mediated SR Ca²⁺ release-dependent and -independent mechanisms may also contribute to impaired VSM function during pathological conditions.

4. MITOCHONDRIAL Ca²⁺ CHANNELS

Intracellular organelles like mitochondria are emerging as important players in smooth muscle Ca²⁺ handling. Mitochondria harbor various Ca²⁺ channels executing mitochondrial Ca²⁺ turnover. Mitochondrial-calcium-uniporter (MCU), mitochondrial RyR, mitochondrial-Ca²⁺-channel type-2, rapid mode of uptake, and H+/Ca²⁺ exchanger (Letm1) play a major role in mitochondrial Ca²⁺ influx. The notable channels for mitochondrial Ca²⁺ extrusion include mitochondrial Na⁺/Ca²⁺ exchanger, mitochondrial permeability transition pores (mPTPs), and Letm1 (which works as an efflux channel at high mitochondrial Ca²⁺ concentrations ([Ca²⁺]_mito)) (Hoppe, 2010). These mitochondrial Ca²⁺ channels may play a physiologically relevant role in VSM, yet they are understudied. In fact, many recent studies centered on the role of the MCU in VSM contractility (McCarron et al., 2013).

In VSM, the mitochondria appear to be a relatively immobile organelle, localized in crucial intracellular regions to operate optimally (McCarron et al., 2013). They sequester cytosolic Ca²⁺ over a wide concentration range (200 nM—10 μM) through the highly Ca²⁺-selective channel MCU. Mitochondrial Ca²⁺ buffering capacity lies in the substantial amount of phosphate inside the organelle and the electrochemical gradient created by expulsion of H⁺ by electron transport chain complexes (McCarron et al., 2013). For instance, mitochondria localized to subplasmalemmal regions of VSM have been shown to buffer stretch-induced cytosolic Ca²⁺ elevation, thereby contributing to intracellular Ca²⁺ homeostasis (Gilbert, Ducret, Marthan, Savineau, & Quignard, 2014). Additionally, evidence suggests that mitochondria do not readily buffer the initial stage of Ca²⁺ influx through LTCC, but rather affect the declining phase of the LTCC-mediated Ca²⁺ signal. However, the organelle is far quicker to scavenge the Ca²⁺ release into the cytosol through IP₃R, thereby targeting the rising phase of Ca²⁺ transient produced by these receptors. Such buffering action may prevent Ca²⁺-dependent deactivation of IP₃R in VSM, thereby allowing repeated occurrence of IP₃R-mediated Ca²⁺ oscillation and Ca²⁺ waves (McCarron et al., 2013). The cue to distinguish the mitochondrial buffering effect on LTCC Ca²⁺ signals vs IP₃R Ca²⁺ signals may lie in a seminal work in neurons that highlights the differential Ca²⁺ sequestering effect of mitochondria on Ca_V1 and Ca_V2channels solely based on the positional/spatial aspect of the organelle with respect to the ion channel (Wheeler et al., 2012). Interestingly, Ca²⁺ uptake by mitochondria does not affect the ATP production by the organelle (Chalmers & McCarron, 2008).

Mitochondria can also regulate VSM function via its production of ROS and modulation of the activity of several ion channels. For example, Ca²⁺ intake by mitochondria residing at close proximity of the IP₃R causes depolarization of the organelle through an elevation in [Ca²⁺]_mito, thereby culminating in increased production of ROS and NF-kB activation (Narayanan, Xi, Pfeffer, & Jaggar, 2010). NF-kB being a transcription modulator may regulate the expression of LTCC, thus influencing arterial contraction (Narayanan et al., 2010). Recent studies have also demonstrated that the vasoconstrictor ANG II couples with NADPH oxidase to produce discrete microdomains of ROS signaling (Amberg, Earley, & Glapa, 2010). These microdomains can be amplified by adjacent mitochondrial ROS-induced ROS release to promote oxidative activation of PKC resulting in local stimulation of LTCC activity, enhanced Ca²⁺ influx and vasoconstriction (Chaplin, Nieves-Cintron, Fresquez, Navedo, & Amberg, 2015). Notably, disruption of this pathway in vivo ameliorates vascular dysfunction associated with hypertension (Chaplin et al., 2015). Mitochondria-derived ROS can also modulate the activity of RyR and BK_Ca channels in VSM and therefore may contribute to vasodilation under certain conditions (Cheranov & Jaggar, 2004; Xi, Cheranov, &Jaggar, 2005). Thus, mitochondria can distinctly regulate VSM function. Future studies should further examine the expression, localization, and function of mitochondrial Ca²⁺ channels, as well as their interplay with other ion channels in modulating cellular Ca²⁺ signals and VSM function.

5. CONCLUSION

Intracellular Ca²⁺ VSM is controlled by an exquisite repertoire of Ca²⁺-permeable channels to regulate cell excitability, vessel diameter, and ultimately, blood flow. Here, we have discussed our current understanding of the expression, structure, localization, regulation, and functional role of major Ca²⁺-permeable channels in VSM. Altered regulation of these Ca²⁺-permeable channels can have profound impact on cardiovascular physiology and pathology. Further research is still required to completely appreciate how all these Ca²⁺-permeable channels contribute to Ca²⁺ handling, VSM excitability, and vascular reactivity. This can be accomplished with the employment of new, emerging technologies. For instance, the development of innovative imaging tools has made possible the recording of sub-cellular Ca²⁺ signals produced by a single or clusters of Ca²⁺-permeable channels. As the superior spatiotemporal resolution afforded by these technologies has begun to refine our understanding of Ca²⁺ signaling, such technologies could be adapted to further examine elementary signals produced by distinct Ca²⁺-permeable channels expressed in VSM. It is increasingly apparent that there is an intricate physical and functional relationship among many of these Ca²⁺-permeable channels as well as with other ion channels and regulatory signaling proteins. Therefore, more comprehensive knowledge of the cellular distribution of these channels with interacting partners is required. The advent of super-resolution nanoscopy as well as proximity ligation assay technology should aid in this task. It will also be important to systematically examine sex- and tissue-specific variations in the expression, localization, regulation, functional role, and physiological significance of all Ca²⁺-permeable channels in VSM. Finally, the role of Ca²⁺-permeable channels in VSM from native human tissue should be examined. This translational approach may confirm mechanisms observed in animal models and, perhaps more importantly, may reveal new information regarding ion channel physiology and pharmacology specific to humans.

ABBREVIATIONS

ANG II

angiotensin II

2-APB

2-aminoethoxydiphenyl borate

BK_Ca

large-conductance Ca²⁺-activated potassium channel

Ca²⁺

calcium

[Ca²⁺]_i

intracellular Ca²⁺ concentration

[Ca²⁺]_mito

mitochondrial Ca²⁺ concentration

CaM

calmodulin

ET-1

endothelin-1

GPCR

G-protein-coupled receptor

IP3R

inositol-1,4,5,-trisphosphate receptor

LTCC

L-type Ca_V1.2 channel

PKA

protein kinase A

PKC

protein kinase C

PKG

protein kinase G

PLC

phospholipase C

ROS

reactive oxygen species

RyR

ryanodine receptor

SM

smooth muscle

SOCE

store-operated Ca²⁺ entry

SR

sarcoplasmic reticulum

STOC

spontaneous transient outward current

TRP

transient receptor potential

TTCC

T-type Ca²⁺ channel

VDCC

voltage-dependent Ca²⁺ channel

VSM

vascular smooth muscle cells