Calcium Signaling in Smooth Muscle

Abstract

Changes in intracellular Ca²⁺ are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca²⁺ signal is a reflection of the source of Ca²⁺ (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca²⁺ entry may be detected optically as graded elevations in intracellular Ca²⁺, junctional Ca²⁺ transients, Ca²⁺ flashes, or Ca²⁺ sparklets, whereas release of Ca²⁺ from intracellular stores may manifest as Ca²⁺ sparks, Ca²⁺ puffs, or Ca²⁺ waves. These diverse Ca²⁺ signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

Ca²⁺ signals in smooth muscle include Ca²⁺ sparks (mediated by RyRs), Ca²⁺ puffs (IP3Rs), Ca²⁺ waves (IP3Rs/RyRs), junctional Ca²⁺ transients (P2X1Rs), Ca²⁺ flashes (VDCCs), and Ca²⁺ sparklets (VDCCs).

SMOOTH MUSCLE

Smooth muscle cells form a continuous layer that lines the walls of the hollow organs of the body, such as blood vessels, intestines, urinary bladder, airways, lymphatics, penis, and uterus. A defining feature of smooth muscle cells is their ability to contract. This property reflects the excitable nature of these cells, which allows for membrane potential-dependent influx of calcium (Ca²⁺) and the Ca²⁺-dependent formation of cross-bridges between myosin and actin—the two major contractile proteins that drive contraction. The contractile property of smooth muscle plays an important functional role in these organs, notably by allowing dynamic changes in luminal volume. These changes may regulate the translational movement of the organ’s contents, such as in the gastrointestinal tract, where the peristaltic action caused by sequential contraction of smooth muscle segments is responsible for the movement of food, and the urinary bladder, where smooth muscle in the wall relaxes during filling and contracts forcefully to expel urine during micturition. Uterine smooth muscle plays a similar role, relaxing during gestation to accommodate fetal growth and contracting vigorously during parturition. In the vasculature, the contractility of smooth muscle in the vessel wall is a primary determinant of blood pressure, which in turn controls blood flow and the distribution of nutrients and oxygen throughout the body.

Ca²⁺ SIGNALS IN SMOOTH MUSCLE

Smooth muscle contractility, and therefore hollow organ function, is regulated by changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). These changes may take a variety of forms, the simplest of which is a “global” (cell-wide) increase of the type associated with Ca²⁺-triggered actin-myosin cross-bridge formation and contraction. However, changes in intracellular Ca²⁺ may also be highly localized and often include a temporal component. Thus, Ca²⁺ changes may be sustained or transient, stationary or moving, or regularly repeating (Berridge 1997; Sanders 2001).

The nature of the Ca²⁺ signal depends to a large extent on the molecular mechanism responsible for generating it. Broadly speaking, there are two major mechanisms by which [Ca²⁺]_i is raised in smooth muscle: (1) entry of Ca²⁺ from the extracellular space, and (2) release of Ca²⁺ from intracellular stores. Influx of extracellular Ca²⁺ is mediated by ion channels in the plasmalemmal membrane, the most prominent of which is the voltage-dependent Ca²⁺ channel (VDCC). Nonselective cation channels, such as transient receptor potential (TRP) channels and ionotropic purinergic (P2X) receptors, are also potentially important extracellular Ca²⁺ entry pathways in smooth muscle cells. Although a number of intracellular organelles take up and release Ca²⁺, the sarcoplasmic reticulum (SR) represents the largest pool of releasable Ca²⁺ in smooth muscle cells. In response to a variety of stimuli, Ca²⁺-release channels in the SR, namely ryanodine receptors (RyR) and inositol trisphosphate receptors (IP₃Rs), mediate efflux of Ca²⁺ from the SR into the cytoplasm of the cell.

IMAGING INTRACELLULAR Ca²⁺

Fluorescence, a property that allows certain molecules to absorb specific wavelengths of light and release energy in the form of light at a longer wavelength, has been exploited to produce numerous fluorescent indicators, including Ca²⁺-sensitive fluorescent dyes. The fluorescence properties of Ca²⁺-sensitive indicators change when Ca²⁺ is bound; thus, such dyes can be used to detect intracellular Ca²⁺ levels. Ion fluxes in smooth muscle can occur very rapidly—often in the millisecond range. This property has motivated the development of a number of very rapid fluorescent dyes that enable detection of such changes in ion concentrations with high temporal resolution.

Fluorescent dyes can be loaded into cells by microinjection, but are more commonly introduced by incubating isolated smooth muscle cells or intact tissue with the membrane-permeant acetoxymethyl (AM) ester of the dye. The AM form is readily taken up by cells, but is acted on by intracellular esterases that cleave the ester bond to release the free anion, which is not membrane permeant and is thus retained within the cell. Most fluorescent Ca²⁺ indicators are based on fluorophore-conjugated derivatives of the Ca²⁺ chelator, BAPTA (bis-[o-amino-phenoxy]-ethane-N,N,N′N′-tetraacetic acid), which is used for both ratiometric and nonratiometric Ca²⁺ applications (Tsien 1980). Ratiometric dyes are used to measure the intracellular concentration of Ca²⁺. These dyes show a shift in the excitation (e.g., Fura-2) or emission (e.g., indole-1) spectrum according to the concentration of free or unbound Ca²⁺. From the ratio of bound and unbound Ca²⁺, the free intracellular Ca²⁺ concentration can be determined. Nonratiometric dyes, such as those in the Fluo family, show an increase in fluorescence quantum yield or intensity on binding Ca²⁺ and are useful for detecting qualitative changes in Ca²⁺ levels. Although nonratiometric dyes are not usually used for quantitative Ca²⁺ measurements, methods have been developed to calculate Ca²⁺ concentrations using single-wavelength fluorescence signals (Jaggar et al. 1998a; Maravall et al. 2000). One advantage of ratiometric dyes is that the ratio normalizes fluorescence variations caused by uneven cell thickness, dye distribution, dye leakage, or photobleaching—problems that are common to nonratiometric dyes. A disadvantage of ratiometric dyes is that they require excitation in the UV range. Some parameters to consider when selecting a fluorescent dye include (1) ion specificity, (2) dissociation constant (K_d), (3) hardware suitability (excitation and emission spectra), (4) fluorescence intensity, (5) availability as an AM ester, and (6) sensitivity to photobleaching.

Ca²⁺ signals are typically imaged using laser-scanning confocal microsopes. In its simplest form, a confocal microscope system comprises three main components: (1) a light source; (2) optical and electronic components to manipulate, display, and analyze signals; and (3) a microscope. Lasers, coupled to either upright or inverted microscopes, are commonly used as the light source for confocal microscopes. Although gas lasers (e.g., Ar-ion, Kr-ion, HeNe) provide numerous lasing lines from UV to red, and are well suited for optimal excitation of fluorescent probes, solid-state lasers are increasingly being used because they offer the advantages of longer lifetime, lower power consumption, and compact size.

To record very rapid events or determine the kinetics of a Ca²⁺ event by confocal microscopy, researchers have typically measured Ca²⁺ fluxes using a line-scanning procedure. In line-scan mode, a single line is repeatedly scanned across the cell for a period of time. Each line is then aligned to form an image that is a plot of fluorescence along the scanned line versus time (Fig. 1). Although line scans are still used, the development of very sensitive CCD (charge-coupled device) cameras and rapid Ca²⁺-sensitive dyes allows laser-scanning confocal systems to routinely achieve detailed spatial and temporal resolution, which is crucial for determining the origin of a Ca²⁺ event.

Figure 1.

Line-scan imaging. The image shown demonstrates the time course of fractional fluorescence (F/F₀; bottom) and spatial distribution of the Ca²⁺ spark (left) fitted to a Gaussian distribution (red line). Gray bar labeled “t” indicates the region over which the fluorescence time course was averaged. Scan lines are displayed vertically in a continuous manner. (Inset) Orientation of scanning line. (Adapted from Bonev et al. 1997; reprinted with permission from The American Physiological Society © 1997.)

Confocal microscopy systems can also be used to measure changes in cellular Ca²⁺ caused by activation of photoprotected (“caged”) compounds. In this technique, the concentration of ions, signaling molecules, or other biologically active compounds can be instantaneously changed by UV stimulation of cells loaded with their caged equivalent. The resulting changes in intracellular Ca²⁺ can be monitored using the same system. By employing caged Ca²⁺ or Ca²⁺-releasing compounds (e.g., IP₃), this approach can also be used to directly elevate Ca²⁺ within a cell. This uncaging strategy facilitates the study of a particular signaling step independent of preceding steps in the signaling pathway.

Specialized techniques have also been developed for measuring Ca²⁺ signals in specific subcellular compartments. One such technique is total internal reflection fluorescence (TIRF) microscopy, which is used to selectively visualize the area immediately beneath the plasmalemma. In TIRF microscopy, an evanescent wave is created by the reflection of a laser beam at the interface between a glass coverslip and the cytoplasm of cells attached to the coverslip (Fig. 2). Because TIRF measurements usually use nonratiometric dyes, they have the same limitations noted above for these fluorophores.

Figure 2.

TIRF microscopy. (A) A schematic of the TIRF imaging system. A, adjustable rectangular knife-blade aperture; BE, beam expander; FL, focusing lens; BF, barrier filter; DM, dichroic mirror; CCD, charge-coupled device. (B) Imaging membrane-proximate fluorescent Ca²⁺ signals near an open channel by TIRF microscopy. (C) A single frame of a TIRF microscopy video image illustrating Ca²⁺ signals generated by three channels within an 80 × 80-μm patch of membrane. Increasing Ca²⁺ concentrations are indicated by both “warmer” colors and height. (Adapted from Demuro and Parker 2004; reprinted with permission from Elsevier © 2004.)

Ca²⁺ SIGNALS FROM OUTER SPACE: Ca²⁺ INFLUX

Signals Mediated by VDCCs

Membrane potential depolarization activates VDCCs—the major contributors to increases in [Ca²⁺]_i. VDCCs are multisubunit complexes comprising a pore-forming α1 subunit and regulatory β, α2δ, and γ subunits (Curtis and Catterall 1984; Hosey et al. 1987; Leung et al. 1987; Vaghy et al. 1987). Most of the functional properties of the VDCC channel, including voltage sensitivity, Ca²⁺ permeability, Ca²⁺-dependent inactivation, and sensitivity to pharmacological block by organic Ca²⁺ channel blockers, are attributable to the α1 subunit. The domain organization of the α1 subunit creates a pseudotetrameric structure in which the four repeat domains (I, II, III, IV), each composed of six transmembrane segments (S1–6) and intracellular amino- and carboxyl-termini (Catterall 2000; Jurkat-Rott and Lehmann-Horn 2004), are analogous to the individual subunits of structurally similar, tetrameric voltage-dependent potassium (K⁺) channels. The S4 transmembrane segments of each domain serve as voltage sensors; in response to changes in membrane potential, they move outward and rotate, producing a conformational change that opens the pore (Catterall 2000).

The pore-forming α1 subunit is expressed as multiple splice variants with different regulatory and biophysical properties. Additional molecular diversity is provided by four different, variably spliced β subunits (Birnbaumer et al. 1998), which further modify VDCC biophysical properties and regulate surface expression of the α1 subunit; properties of the VDCC complex may be additionally modulated by splice variants of the α2δ regulatory subunit (Angelotti and Hofmann 1996).

VDCC-mediated currents are characterized by high voltage of activation, large single-channel conductance, and slow voltage-dependent inactivation. VDCC-mediated currents also display a characteristic sensitivity to dihydropyridines (Reuter 1983), a class of drugs used clinically in the treatment of hypertension (Nelson et al. 1990; Snutch et al. 2001). In vascular and visceral smooth muscle, these dihydropyridine-sensitive currents are attributable to the expression of the L-type VDCC pore-forming α1C subunit (Ca_V1.2) (Keef et al. 2001; Moosmang et al. 2003; Wegener et al. 2004); but in some smooth muscle types, the α1D (Ca_V1.3) subunit is expressed as well (Nikitina et al. 2007). Of the four β subunits, only two—β2 and β3—are clearly detected at the protein level in smooth muscle, although mRNA for all four isoforms is expressed (Hullin et al. 1992; Murakami et al. 2003).

There is also evidence for the expression of a dihydropyridine-insensitive Ca²⁺ current in some smooth muscle types. This T-type (transient) current is mediated by Ca_V3 pore-forming α subunits—primarily the Ca_V3.1 isoform in smooth muscle (Bielefeldt 1999; Perez-Reyes 2003). Expression of T-type channels varies between different smooth muscle types, but their presence often goes undetected because of their low levels of expression or because they are obscured by the specific recording conditions used. Moreover, the negative steady-state inactivation property of these channels results in their being half-inactivated at about –70 mV. Therefore, over the range of smooth muscle resting membrane potential (–50 to –30 mV), T-type channels may be completely inactivated. Because of these gating properties, the importance of T-type currents in the regulation of smooth muscle membrane potential is a matter of controversy. However, there is evidence from the rabbit urethra that, at least in this tissue, T-type channels regulate action potential frequency (Bradley et al. 2004). It also has been suggested that voltage-dependent Ca²⁺ currents in smooth muscle with a T-type pharmacology may arise because of a Ca_V3.1 (and/or Ca_V3.2) splice variant with a more depolarized activation voltage (Kuo et al. 2010).

Three different Ca²⁺ signals mediated by VDCCs have been identified in smooth muscle: (1) global elevations in intracellular Ca²⁺, (2) Ca²⁺ “flashes,” and (3) Ca²⁺ “sparklets.”

Global Ca²⁺ Signals

As the name suggests, global Ca²⁺ signals reflect changes in Ca²⁺ concentration that are essentially uniform throughout the cell. In smooth muscle, depolarization of the membrane by ∼15 mV from its resting potential (approximately –50 to –40 mV) elevates global Ca²⁺ to ∼300–400 nM, whereas hyperpolarization by ∼15 mV lowers Ca²⁺ to ∼100 nM. These signals are typically monitored using ratiometric dyes (e.g., Fura-2), which are ideally suited to measuring Ca²⁺ concentration over this range (K_d ∼300 nM). Thus, by modulating the steady-state open probability of VDCCs, slow changes in membrane potential can have profound, sustained effects on global intracellular Ca²⁺. This is illustrated in Figure 3, which shows that the membrane potential depolarization that accompanies elevation of intraluminal pressure from 60 mmHg (Fig. 3A) to 100 mmHg (Fig. 3B) increases the global intracellular Ca²⁺ concentration in the vascular wall. Inhibition of VDCC channels with nisoldipine decreases global [Ca²⁺]_i (Fig. 3C). Changes in global Ca²⁺ are accompanied by changes in vascular diameter, underscoring the importance of global Ca²⁺ in regulating the contractile state of smooth muscle (see below).

Figure 3.

Global Ca²⁺. Ca²⁺ images obtained from a rat basilar artery pressurized to (A) 60 mmHg, (B) 100 mmHg, and (C) 100 mmHg in the presence of nisoldipine. The numbers below each panel correspond to the Ca²⁺ concentration in the smooth muscle of the vascular wall, calculated from the ratio of Fura-2 fluorescence at 340 and 380 nm. Note contraction and dilation in B and C, respectively, relative to A. (Adapted from Knot and Nelson 1998; reprinted with permission from The Journal of Physiology © 1998.)

Ca²⁺ Flashes

Some types of smooth muscle (e.g., urinary bladder, gallbladder, ureter) show action potentials. These rapid, transient changes in membrane potential, which show a characteristic temporal profile, are unique to excitable cells. However, unlike other excitable cells, such as neurons and cardiac myocytes, where action potentials are initiated by activation of channels that predominantly mediate sodium (Na⁺) influx, the upstroke of the action potential in smooth muscle reflects massive Ca²⁺ entry through VDCCs. The resulting rapid elevation of global intracellular Ca²⁺ can be detected optically as a Ca²⁺ flash that brightly and briefly lights up cells loaded with fluorescent Ca²⁺ indicators. A recording of a Ca²⁺ flash in urinary bladder smooth muscle is shown in Figure 4. Ca²⁺ flashes can be evoked by electrical field stimulation, but also occur spontaneously (as in the example shown), likely reflecting the spontaneous release of neurotransmitters from sympathetic (e.g., mesenteric arteries) or parasympathetic (e.g., urinary bladder) nerve terminals (Klockner and Isenberg 1985; Heppner et al. 2005). Note that in this example, Ca²⁺ was simultaneously elevated in two adjacent cells, indicating that these events may be coupled.

Figure 4.

Ca²⁺ flashes. (A) Selected images recorded before (a), during (b), and after (c) a spontaneous action potential in a smooth muscle bundle loaded with Fluo-4 and impaled with a microelectrode (green rectangle). (B) Simultaneous recordings of changes in Ca²⁺-activated fluorescence (upper trace) and voltage (lower trace) from a single bundle of urinary bladder smooth muscle. Changes in Ca²⁺-activated fluorescence were measured from the red box in A; the letters a, b, and c denote the times at which the correspondingly labeled images in A were acquired. Note that each of the three action potentials induced a simultaneous increase in Ca²⁺-activated fluorescence. (Adapted from Heppner et al. 2005; reprinted with permission from The Journal of Physiology © 2005.)

Ca²⁺ Sparklets

Cheng and colleagues first measured the local Ca²⁺ signal caused by the opening of a single L-type VDCC in cardiac muscle (Wang et al. 2001b), and referred to these events as Ca²⁺ sparklets. Ca²⁺ sparklets are also present in vascular smooth muscle (Fig. 5), where they are detected by TIRF microscopy as highly localized, dihydropyridine-sensitive, subplasmalemmal Ca²⁺-release events reflecting the activity of an individual channel or cluster of channels (Navedo et al. 2005). The average area of a Ca²⁺ sparklet is ∼0.8 µm² or ∼0.08% of the surface membrane of a typical arterial smooth muscle cell (Santana et al. 2008). Ca²⁺ influx through Ca²⁺ sparklet sites is quantal, and the size of a given Ca²⁺ sparklet depends on the number of quanta activated. One quantal unit of Ca²⁺ release elevates [Ca²⁺]_i by about 35 nm. The specific locations of Ca²⁺ sparklets vary between cells, but within a cell, Ca²⁺ sparklets are predominantly stationary events that occur in specific regions of the sarcolemmal membrane (Fig. 5A–C). Both low- and high-activity Ca²⁺ sparklet sites have been described. The frequency of the latter type of event, termed a persistent sparklet, is increased by activation of protein kinase C (PKC), which recruits previously silent sites and increases the frequency of low-activity sites (Fig. 5D).

Figure 5.

Ca²⁺ sparklets. (A) Surface plot of Ca²⁺ imaged in a freshly isolated arterial myocyte. (Inset) Higher magnification view of boxed area showing three active Ca²⁺ sparklet sites (2 mm extracellular Ca²⁺). (B) Traces showing time course of changes in Ca²⁺ at sites a–d. (C) Amplitude histogram of Ca²⁺ sparklets (20 mm extracellular Ca²⁺). (D) Amplitude histogram of Ca²⁺ sparklets (2 mm extracellular Ca²⁺) in the absence and presence of the PKC activator phorbol 12,13-dibutyrate (PDBu). Solid lines in C and D are best fits to a Gaussian function. (Adapted from Navedo et al. 2005; reprinted with permission from The National Academy of Sciences © 2005.)

In heart muscle, where local coupling of Ca²⁺ influx through single VDCCs to RyRs is central to excitation-contraction coupling (Cannell et al. 1995; Lopez-Lopez et al. 1995), a Ca²⁺ sparklet can activate four to six nearby RyRs to cause a Ca²⁺ spark (see below). However, there is no evidence for this direct VDCC-to-RyR communication in smooth muscle. Instead, Ca²⁺ currents through VDCCs appear to activate RyRs indirectly through elevations in global Ca²⁺ and SR Ca²⁺ load (Collier et al. 2000; Herrera and Nelson 2002; Wellman and Nelson 2003; Essin and Gollasch 2009).

Signals Mediated by Store-Operated Ca²⁺ Channels

Extracellular Ca²⁺ influx in response to depletion of intracellular Ca²⁺ stores, a process termed store-operated Ca²⁺ entry (SOCE), is known to play an important role in a number of cell types, notably nonexcitable cells. However, the molecular mechanism underlying this coupling long remained elusive. Recent seminal work by a number of independent groups effectively resolved this question, clearly identifying ubiquitously expressed STIM proteins (Liou et al. 2005; Roos et al. 2005) as endoplasmic reticulum (ER) Ca²⁺ sensors, and members of the Orai family (Feske et al. 2006; Vig et al. 2006; Zhang et al. 2006) of transmembrane proteins as the entities responsible for mediating Ca²⁺ entry (reviewed in Varnai et al. 2009). These researchers showed that, in response to a decrease in ER Ca²⁺ concentrations, the low-affinity Ca²⁺-binding STIM proteins aggregate to form discrete plasmalemmal-proximate clusters that tether Orai proteins. This physical coupling activates Orai, which is a highly selective Ca²⁺ channel, thereby promoting extracellular Ca²⁺ entry. The identification of the STIM-Orai mechanism has sparked renewed interest in investigating SOCE in smooth muscle (reviewed in Wang et al. 2008). These studies, most of which have been performed using cultured smooth muscle cells, have consistently shown that STIM and Orai family members are expressed in smooth muscle, and, under the conditions tested, are capable of functionally coupling store depletion to extracellular Ca²⁺ entry (Peel et al. 2006, 2008; Takahashi et al. 2007; Ng et al. 2010; Park Hopson et al. 2011). It has also been suggested that, in addition to promoting Orai activity, STIM1 negatively regulates VDCCs (Wang et al. 2010). Some recent studies have provided evidence for STIM-Orai coupling in native smooth muscle preparations, and have suggested a role for this mechanism in hypertension (Giachini et al. 2009, 2010). An optical signature of Orai-mediated Ca²⁺ influx has not been defined and additional research will be required to definitively establish the physiological relevance of this pathway in native smooth muscle tissues.

Signals Mediated by Nonselective Cation Channels

In contrast to VDCCs, which show a high selectivity for Ca²⁺ ions over monovalent cations, nonselective cation channels typically also allow influx of extracellular Na⁺. Although channels of this type are permeable to Ca²⁺ and thus directly increase [Ca²⁺]_i to some degree, in many if not most cases, their major impact on [Ca²⁺]_i is indirect through Na⁺-dependent membrane potential depolarization and activation of VDCCs. Several types of Ca²⁺-permeable, nonselective cation channels are present in smooth muscle, including receptor-activated channels, mechanosensitive channels, tonically active channels, and channels activated by SR Ca²⁺ store depletion.

Receptor-Activated Cation Channels

Of the various nonselective cation channels expressed in smooth muscle, only the ATP-gated P2X receptor (P2XR) is clearly associated with an optically identifiable Ca²⁺ signal. The functional P2XR complex is thought to be a trimer (Aschrafi et al. 2004)—an unusual structural arrangement in ion-channel space where tetramers dominate. Each subunit contains intracellular amino- and carboxyl-termini and two membrane-spanning domains that are connected by a large extracellular domain (Khakh 2001; North 2002). Binding of ATP (three molecules per complex) to a site in the large extracellular domain induces a conformational change that results in rapid (milliseconds) opening of the pore.

These Ca²⁺- and Na⁺-permeable channels (Benham and Tsien 1987; Schneider et al. 1991) mediate a rapid local influx of Na⁺ and Ca²⁺ at nerve–muscle junctions following activation by neurally released ATP (Lamont and Wier 2002; Lamont et al. 2006). The influx of Na⁺ and Ca²⁺ creates an excitatory junction potential (EJP) that contributes directly to the increase in postjunctional excitability. Multiple lines of evidence, including studies using knockout mice, indicate that the P2X₁ receptor is the predominant P2X receptor isoform expressed in smooth muscle (Mulryan et al. 2000; Vial and Evans 2002; Lamont et al. 2006; Heppner et al. 2009).

Although most of the excitatory junction current (EJC) associated with P2X₁ receptor activation is carried by the more abundant Na⁺ ions, Ca²⁺ influx is substantial. This influx can be detected optically in the form of local elementary purinergic-induced Ca²⁺ transients (Fig. 6). These events have been described in vas deferens (Brain et al. 2002), mesenteric arteries (Lamont and Wier 2002), and urinary bladder (Heppner et al. 2005), where they have been termed neuroeffector Ca²⁺ transients (NCTs), junctional Ca²⁺ transients (jCaTs), and nerve-evoked elementary purinergic Ca²⁺ transients, respectively. The kinetic properties of these purinergic Ca²⁺ signals are similar to one another and are clearly distinct from those of other local Ca²⁺ transients (Hill-Eubanks et al. 2010).

Figure 6.

Elementary purinergic Ca²⁺ transients recorded from urinary bladder smooth muscle. Transient events were evoked by electrical field stimulation (2-sec train, 5 Hz; 37°C) in bladder strips loaded with Fluo-4 and scanned at a rate of 30 images/sec. The top three images illustrate nerve processes before (top panel, left, indicated by arrows) and after stimulation (middle and right panels). The three bottom panels illustrate color-coded ratios (F/F₀) of the images above. Intracellular Ca²⁺ increases first in the nerve fibers, and then two local Ca²⁺ transients are detected (right panel, bottom right quadrant, indicated by arrows). The activity of Ca²⁺ transients continued throughout the duration of field stimulation. (Unpublished data from Mark Nelson.)

Other Nonselective Cation Channels

Smooth muscle cells express a number of nonselective cation channels of the TRP family. Although no signature signaling event associated with Ca²⁺ influx through TRP channels has been reported in the literature, given the relative selectivity of these channels for Ca²⁺ (and the high Ca²⁺ permeability and single-channel conductance of some TRP family members, notably TRPV), imaging methods that have been used to examine jCaT-like events (confocal microscopy) and/or VDCC-mediated sparklets (TIRF) may ultimately provide a means to optically detect Ca²⁺ influx through these channels.

Ca²⁺ SIGNALS FROM INNER SPACE: RELEASE OF Ca²⁺ FROM INTRACELLULAR STORES

The most important intracellular Ca²⁺ store in smooth muscle is the SR. Cytosolic Ca²⁺ is transported into the SR by the action of the SR/ER Ca²⁺ ATPase (SERCA). SERCA activity is negatively regulated by the protein phospholamban, a target of protein kinase A (PKA) and protein kinase G (PKG). On phosphorylation, the SERCA inhibitory activity of phospholamban is lost, increasing Ca²⁺ uptake and SR Ca²⁺ load. Free Ca²⁺ taken up by the SR is buffered by Ca²⁺-binding proteins, which retain transported Ca²⁺ and reduce the free Ca²⁺ concentration gradient, facilitating continued Ca²⁺ uptake from the cytoplasm (Pozzan et al. 1994).

Ca²⁺ sequestered in the SR may be delivered to the cytosol through RyRs and IP₃ receptors in the SR membrane. Although these two channel types are phenotypically similar on a superficial level (both function to release Ca²⁺ from SR stores) they are very different molecular entities with distinctive regulatory features and characteristic Ca²⁺-release signatures.

Signals Mediated by RyRs: Ca²⁺ Sparks

There are three RyR subtypes (RyR1-3), each of which is expressed at varying levels in different smooth muscle tissues (Neylon et al. 1995; Yang et al. 2005; Prinz and Diener 2008). RyRs are large tetrameric complexes formed from ∼560-kDa subunits. Each subunit contains four membrane-spanning domains, a large cytosol-facing amino-terminal region containing the Ca²⁺-binding site as well as binding sites for numerous accessory proteins, and a short SR-luminal carboxy-terminal domain (reviewed in Zalk et al. 2007; Lanner et al. 2010).

Ca²⁺ flux through ryanodine receptors is detectable in the form of elementary release events termed Ca²⁺ sparks. First discovered in cardiac muscle (Cheng et al. 1993) and subsequently identified in skeletal (Klein et al. 1996) and smooth (Nelson et al. 1995) muscle, a Ca²⁺ spark represents the opening of a few (likely four to six) RyR channels in the SR membrane (Cheng and Lederer 2008). Ca²⁺ sparks have been detected in a wide variety of smooth muscle types, including those from arteries (Nelson et al. 1995), portal vein (Mironneau et al. 1996; Gordienko and Bolton 2002), urinary bladder (Herrera et al. 2001), ureter (Burdyga and Wray 2005), airway (Sieck et al. 1997), and the gastrointestinal tract (Gordienko et al. 1998). Ca²⁺ sparks in arterial smooth muscle can be detected in isolated myocytes as well as in intact pressurized arteries. Ca²⁺ sparks are rapid, transient, stationary events. The rise time of sparks in vascular smooth muscle and urinary bladder smooth muscle is ∼20–40 msec (Nelson et al. 1995; Herrera et al. 2001); their spatial spread is ∼12.6 µm, and this spread corresponds to ∼1% of the surface membrane. The duration (half-time) of these events is ∼50–60 msec; this contrasts with nerve-evoked purinergic Ca²⁺ transients, which have half-times of ∼110–145 msec (Brain et al. 2002; Lamont and Wier 2002; Heppner et al. 2005). Current evidence indicates that smooth muscle Ca²⁺ sparks are attributable to activation of RyR2, although both RyR1 and RyR3 may influence spark activity (Vaithianathan et al. 2010).

In smooth muscle, localized increases in Ca²⁺ associated with Ca²⁺ sparks activate closely juxtaposed large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channels in the plasma membrane. The K_d of BK_Ca channels for Ca²⁺ is ∼20 µm at the physiological membrane potential of –40 mV. A single spark causes a local increase of [Ca²⁺]_i of 10–30 µm and activates about 30 nearby BK_Ca channels, increasing their open probability by approximately 100-fold (Jaggar et al. 2000; Perez et al. 2001). In smooth muscle from adult animals, there is a one-to-one relationship between sparks and BK_Ca channel–mediated transient outward currents, indicating that all spark sites are functionally coupled to BK_Ca channel clusters. In current-clamp mode, activation of BK_Ca channels by a single spark causes about a 20-mV hyperpolarization (Jaggar et al. 1998b).

Stimuli that increase SR Ca²⁺ load increase the frequency of Ca²⁺ sparks, but sparks also occur spontaneously. The outward currents associated with this latter activity are termed “spontaneous transient outward currents” or “STOCs” (Benham and Bolton 1986). An example depicting the time course and decay kinetics of a single Ca²⁺ spark event is presented in Figure 7A. Simultaneous electrophysiological recordings and traces showing analyzed Ca²⁺ signals (Fig. 7B) highlight the one-to-one relationship between sparks and STOCs.

Figure 7.

Ca²⁺ sparks in an isolated rat basilar artery myocyte. (A) (Top) Two-dimensional confocal images of an entire smooth muscle cell showing the time course of the fractional increase in Fluo-3 fluorescence (F/F₀) of a typical Ca²⁺ spark. (Bottom) Images obtained from the region of interest in the top-right panel (dotted box) depicting spark decay. Pseudocolor denotes relative Ca²⁺ levels as indicated by the bar. (B) Simultaneous measurements of STOCs and sparks at –40 mV highlighting the temporal association between the events. The pink bar below the sparks trace corresponds to the time period imaged in A. (Adapted from Perez et al. 1999; reprinted with permission from The Rockefeller University Press © 1999.)

In addition to activating BK_Ca channels to produce STOCs, Ca²⁺ sparks can also activate Ca²⁺-sensitive chloride (Cl_Ca) channels to produce spontaneous transient inward currents (STICs) (Hogg et al. 1993). Where BK_Ca and Cl_Ca channels coexist, spontaneous transient outward/inward currents (STOICs) are produced (ZhuGe et al. 1998; Jaggar et al. 2000; Wellman and Nelson 2003). Both hyperpolarizing (STOCs) and depolarizing currents (STICs) modulate membrane potential and excitability, with STOCs being inhibitory and STICs being excitatory.

Signals Mediated by IP₃Rs: Ca²⁺ Waves

There are three IP₃ receptor subtypes (IP₃R1-3), each of which is expressed at varying levels in different smooth muscle tissues (Newton et al. 1994; Tasker et al. 1999; Boittin et al. 2000; Grayson et al. 2004). IP₃Rs are large homo- or heterotetrameric complexes formed from approximately 2700 to 2800 amino acid subunits. Each subunit contains six membrane-spanning domains, a large cytosol-facing amino-terminal region containing the IP₃ binding site, and a short cytosolic carboxy-terminal domain. The pore of the channel is formed by the coassociation of transmembrane domains 5 and 6 from each of the four subunits (Foskett et al. 2007).

Ca²⁺ release by IP₃Rs is regulated by two second messengers: IP₃ and Ca²⁺. The prototypical signaling pathway that leads to elevation of IP₃ is activation of Gα_q/11-type G protein-coupled receptors. Among the most prominent agonists of this pathway in smooth muscle are neurohumoral vasoconstrictors. Activation of this pathway stimulates phospholipase C, resulting in hydrolysis of membrane-associated phosphoinositide 4,5-bisphosphate (PIP₂) into IP₃ and diacyclglycerol. The IP₃ generated by this pathway binds IP₃Rs and promotes channel gating, releasing Ca²⁺ into the cytosol. The Ca²⁺ released by IP₃Rs can reciprocally modulate IP₃R activity in two ways: as Ca²⁺ rises from low nanomolar basal levels to low micromolar levels in the vicinity of the channel, it activates IP₃Rs; at higher local levels, the channel becomes inactivated. These activation/inactivation properties together with regulation of channel function by multiple interacting factors, allow IP₃Rs to generate a large variety of temporally and spatially modulated Ca²⁺-signaling patterns within the cell (Foskett et al. 2007).

In some smooth muscle types, IP₃R-mediated Ca²⁺ release is transient and localized. For example, smooth muscle cells from colon and portal vein show spontaneous Ca²⁺ spark-like events that are enhanced by IP₃ production and eliminated by IP₃R blockade (Bayguinov et al. 2000; Gordienko and Bolton 2002). These Ca²⁺ release events, termed Ca²⁺ “puffs,” have a biophysical signature (e.g., kinetics, magnitude, spatial spread) that distinguishes them from the RyR-mediated Ca²⁺ sparks that are prominent in most other smooth muscle types (e.g., vascular smooth muscle, gallbladder, and urinary bladder) (Nelson et al. 1995; Herrera et al. 2001; Pozo et al. 2002). Although Ca²⁺ puffs are unitary events, they can act as initiation sites for intracellular Ca²⁺ waves and thereby contribute to global Ca²⁺ signals (Bootman and Berridge 1996; Thomas et al. 1998).

A Ca²⁺ wave, defined as an increase in [Ca²⁺]_i that propagates across the entire smooth muscle cell from an initial site of release, is perhaps the most studied IP₃R-mediated Ca²⁺-signaling event. First described by Iino in rat tail arteries (Fig. 8), Ca²⁺ waves are a common feature of vascular smooth muscle cells exposed to Gα_q/11-coupled vasoconstrictor agonists, such as UTP (Jaggar and Nelson 2000) and norepinephrine (Iino et al. 1994; Boittin et al. 1999; Miriel et al. 1999; Ruehlmann et al. 2000), or electrical field stimulation of perivascular nerves.

Figure 8.

Ca²⁺ waves. (A) Two-dimensional pseudocolor confocal microscopic images of rat tail artery smooth muscle showing dynamic, recurrent changes in intracellular Ca²⁺ (measured by changes in the intensity of Fluo-3 fluorescence) following electrical stimulation of perivascular sympathetic nerves. Six of 96 consecutive frames collected at a rate of one frame per second are shown. (B) Red, blue, and green lines depict changes in fluorescence intensity as a function of time (a–f in A) in three selected regions of interest (white boxes in a). (Adapted from Iino et al. 1994; reprinted with permission from The EMBO Journal © 1994.)

In the current view, Ca²⁺ waves reflect the activation/inactivation properties of IP₃Rs, and arise through a regenerative Ca²⁺-induced Ca²⁺ release (CICR) mechanism. Ca²⁺ released from the SR acts on successive adjacent IP₃Rs or IP₃R clusters in a cascading fashion, creating a leading edge of Ca²⁺ elevation that traverses the length of the cell. When Ca²⁺ waves propagate toward each other and collide, they cancel each other out because of depletion of SR Ca²⁺ stores on either side of the collision site (Stevens et al. 1999). Therefore, a continuous cycling of SR Ca²⁺ release and reuptake maintains the Ca²⁺ waveform. Ca²⁺ released from SR stores is sufficient for further release and maintenance of Ca²⁺ waves, indicating that the mechanism of smooth muscle Ca²⁺ wave propagation is independent of extracellular Ca²⁺ entry (Boittin et al. 1999; Jaggar and Nelson 2000; Peng et al. 2001; Heppner et al. 2002). Repeating IP₃R-dependent Ca²⁺ signals may also take the form of whole-cell oscillations in [Ca²⁺]_i.

The view of Ca²⁺ waves as strictly IP₃R-mediated events oversimplifies the true situation. In actuality, Ca²⁺ waves arise in response to activation of IP₃Rs and/or RyRs (Iino et al. 1994; Boittin et al. 1999; Hirose et al. 1999; Jaggar and Nelson 2000; Lee et al. 2002), and their properties as well as the relative contributions of IP₃Rs and RyRs may differ depending on the nature of the stimulus or tissue context. In some cases, these events appear to exclusively reflect the activity of RyRs. One such example is provided by rat cerebral arteries, where Nelson and colleagues (Heppner et al. 2002) have found that Ca²⁺ waves are induced by caffeine, which acts on RyRs but not IP₃Rs. Moreover, these events are insensitive to the nominally selective IP₃R blockers, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB), but are completely eliminated by ryanodine. Interestingly, RyR-mediated Ca²⁺ signaling in this preparation is sharply dependent on pH. Increasing the pH of the bathing solution from 7.4 to 7.5 increased Ca²⁺ spark frequency by ∼50%; above this pH, Ca²⁺ waves came to predominate, increasing approximately threefold between pH 7.5 and pH 7.6 and doubling again at pH 7.7–7.8.

FUNCTIONAL CORRELATES OF Ca²⁺ SIGNALS

Contractility

Smooth muscle contraction is driven by Ca²⁺-calmodulin activation of myosin light chain kinase, which has a Ca²⁺ half-activation of ∼400 nm (Stull et al. 1998). The gain of smooth muscle contraction to Ca²⁺ can be adjusted through regulation of myosin light chain phosphatase (Somlyo and Somlyo 2003; Mizuno et al. 2008).

Global Ca²⁺

Membrane potential plays an important role in all excitable cells, including smooth muscle, where it regulates [Ca²⁺]_i and thereby smooth muscle contraction. The resting membrane potential of smooth muscle is approximately –50 to –40 mV, which is positive to the equilibrium potential for K⁺ (E_K). In arterial smooth muscle, this membrane potential is sufficient to increase the steady-state open probability of VDCCs, elevate global intracellular Ca²⁺ from ∼100 nm to ∼200 nm, and cause a tonic constriction (Knot and Nelson 1998). As noted above, membrane potential hyperpolarization to –60 mV lowers Ca²⁺ to about 100 nm, and depolarization to about –30 mV elevates global Ca²⁺ to ∼300–400 nm; these changes in global intracellular Ca²⁺ are sufficient to cause maximal dilation and constriction, respectively. The fundamental relationship between global Ca²⁺ and smooth muscle membrane potential is depicted in Figure 9, which also highlights the role of intravascular pressure as a physiological driver of changes in membrane potential. Oscillations in membrane potential caused by fluctuations in Ca²⁺ entry through VDCCs lead to vasomotion of the arterial wall.

Figure 9.

Intravascular pressure-membrane potential-[Ca²⁺]_i relationships. Fundamental relationships among intravascular pressure (P10–P100, in mm Hg), membrane potential, and arterial wall Ca²⁺. (Adapted from Knot and Nelson 1998; reprinted with permission from The Journal of Physiology © 1998.)

Junctional Ca²⁺ Transients and Ca²⁺ Flashes

Nerve stimulation–evoked localized Ca²⁺ influx through P2X₁R channels causes a depolarizing current carried by Na⁺ and Ca²⁺ ions that activates VDCCs; in urinary bladder smooth muscle, this manifests as a Ca²⁺ flash. Coordinated flash activity among smooth muscle cells in a bundle leads to a transient contraction. Thus, junctional Ca²⁺ transients mediated by P2X₁Rs may indirectly modulate contraction by triggering VDCC activity.

The bursts of local elevations in intracellular Ca²⁺ provided by junctional Ca²⁺ transients also have the potential to trigger activation of proximate RyRs in the SR through a CICR mechanism. The best evidence for the existence of such a mechanism comes from studies of the vas deferens by Cunnane and coworkers (Brain et al. 2003). In this preparation, the magnitude of neurally evoked, ATP-induced Ca²⁺ transients was reduced by ∼45% by inhibition of RyRs with ryanodine. Moreover, treatment with caffeine to increase RyR activity produced a 16-fold increase in the frequency of neurally evoked junctional Ca²⁺ transients. Collectively, these results argue that the neurally evoked Ca²⁺ signal associated with extracellular Ca²⁺ influx triggers—and merges with—a RyR-mediated Ca²⁺ signal, creating an optically detectable signal that reflects a summation of the two separate transient release events. The more modest inhibitory effect of ryanodine on jCaTs in mesenteric arteries (∼13%) (Lamont and Wier 2002) and the apparent absence of an effect of ryanodine on purinergic Ca²⁺ transients in rat urinary bladder (Heppner et al. 2005) suggest a degree of variability among tissues and species. How (or if) this communication from P2X₁Rs to RyRs influences the contractile behavior of smooth muscle is not clear. The additional increment of Ca²⁺ may sum with P2X₁R and VDCC-mediated Ca²⁺ to augment the transient contraction. Alternatively, CICR-activated RyRs could, in theory, couple to BK_Ca channels to oppose contraction. Depending on the relative speed of IP₃ production by concurrent activation of adrenergic or muscarinic receptors, it is also possible that Ca²⁺ influx through P2X₁Rs could amplify local IP₃R activation by IP₃, a possibility that has not yet been explored experimentally.

Ca²⁺ Sparks

In cardiac and skeletal muscle, local Ca²⁺ entry through VDCCs activates proximate RyRs, producing Ca²⁺ sparks that summate to create a substantial increase in global Ca²⁺; thus, Ca²⁺ sparks play a dominant role in contraction in these tissues. In smooth muscle, the molecular architecture is much different, resulting in unique linkages that create a phenotypically opposite functional outcome. In particular, the close physical coupling between VDCCs and RyRs that characterizes striated muscle cells is absent in smooth muscle cells. In its place is a close linkage between the RyR and plasma membrane BK_Ca channels, which are not expressed in cardiac or skeletal muscle cells. As a result of this unique architecture, Ca²⁺ released by RyRs in the SR in the form of sparks activates juxtaposed BK_Ca channels, promoting an outward K⁺ current that hyperpolarizes the smooth muscle membrane and reduces VDCC activity. The resulting decrease in Ca²⁺ influx thus opposes VDCC-mediated smooth muscle contraction (Nelson et al. 1995; Perez et al. 1999; Jaggar et al. 2000). Under this scenario, Ca²⁺ influx through VDCCs initiates the BK_Ca channel-mediated feedback mechanism by enhancing RyR activity, by increasing global Ca²⁺ and SR Ca²⁺ stores (Collier et al. 2000; Herrera and Nelson 2002; Wellman and Nelson 2003; Essin and Gollasch 2009).

As is observed with P2X₁ agonists, simultaneous activation of RyRs by rapid addition of high levels of the RyR activator, caffeine, can cause global Ca²⁺ transients and a transient contraction (Wellman and Nelson 2003).

Ca²⁺ Waves

Ca²⁺ waves normally occur asynchronously in smooth muscle cells. However, in some vascular beds, Ca²⁺ waves may synchronize in neighboring arterial myocytes to initiate vasomotion (Peng et al. 2001), supporting the idea that Ca²⁺ waves can supply the Ca²⁺ needed for smooth muscle contraction (Kasai et al. 1997; Boittin et al. 1999; Mufti et al. 2010). Alternatively, Ca²⁺ waves can influence contractility indirectly through activation of Ca²⁺-dependent ion channels located in the plasmalemmal membrane. For example, Ca²⁺ waves can activate Cl_Ca channels to promote membrane depolarization leading to enhanced Ca²⁺ entry through VDCCs (Mironneau et al. 1996). Ca²⁺ waves can also activate BK_Ca channels, thereby promoting membrane potential hyperpolarization, closure of VDCCs, and induction of smooth muscle relaxation (Young et al. 2001). Thus, while information on Ca²⁺ waves in smooth muscle continues to accumulate, the physiological function of these Ca²⁺ signals remains uncertain.

Ca²⁺-DEPENDENT TRANSCRIPTION FACTOR ACTIVATION

Global Ca²⁺: Excitation-Transcription Coupling

Excitation-contraction coupling, in which depolarization induces contraction through VDCC-mediated increases in intracellular Ca²⁺, is paralleled by a conceptually similar mechanism that links depolarization-induced increases in [Ca²⁺]_i to activation of Ca²⁺-sensitive transcription factors. This process, which has been termed excitation-transcription coupling, translates short-term Ca²⁺-signaling and contractile events into long-term regulation of the smooth muscle cell transcriptome. Unlike cardiac and skeletal muscle cells, smooth muscle cells are highly plastic; their phenotype is maintained through dynamic regulation of gene expression in response to environmental cues (Owens 1995). Thus, excitation-transcription coupling serves to maintain the contractile phenotype by promoting the expression of smooth muscle-specific genes. It also provides a mechanism for phenotypic switching to a “synthetic” phenotype characterized by expression of genes that promote proliferation, matrix deposition, and other functions that come into play under pathological conditions and in the context of vessel repair and new vessel formation. Recent work from Owens and colleagues (Wamhoff et al. 2004) has shown that VDCC-mediated elevations in Ca²⁺ act through two distinct mechanisms to regulate the contractile and synthetic/proliferative phenotypes. In the first, depolarization-induced Ca²⁺ elevation induces SRF (serum response factor)-regulated smooth muscle–specific genes (e.g., myosin heavy chain, smooth muscle α-actin) through activation of Rho/Rho kinase and stimulation of myocardin, a potent coactivator of SRF first identified by Olson and colleagues (Wang et al. 2001a). In the second mechanism, elevated intracellular Ca²⁺ acts through calmodulin-dependent kinase (CaMK) to activate CREB (cAMP responsive element binding protein) and the immediately early gene, c-fos, which is involved in proliferative responses. A similar CaMK/CREB-dependent mechanism has been implicated in the VDCC-mediated induction of Egr-1 (Pulver-Kaste et al. 2006) and c-fos (Cartin et al. 2000) in native cerebral arteries, and TRP channels in gall bladder smooth muscle (Morales et al. 2007). In this latter study, a role for the phosphatase calcineurin was also suggested.

Spatially and Temporally Modulated Ca²⁺ Signals

Studies on the effects of Ca²⁺ signal modulation on transcription factor activation in smooth muscle cells are limited, but seminal work by Lewis and Tsien and colleagues (Dolmetsch et al. 1998; Li et al. 1998) in nonexcitable cells has shown a role for amplitude and frequency modulation of Ca²⁺ signals in differentially regulating the activity of Ca²⁺-sensitive transcription factors. These researchers showed that large transient increases in [Ca²⁺]_i are sufficient to robustly activate NF-κB and c-Jun terminal kinase (JNK) but not NFAT (nuclear factor of activated T-cells), which is effectively activated by a sustained, graded increase in global intracellular Ca²⁺. It has been further shown that activation of Ca²⁺-sensitive transcription factors is modulated by oscillatory elevations in intracellular Ca²⁺: for all transcription factors tested (NFAT, Oct/OAP, and NF-κB), high-frequency oscillations enhanced the efficacy of a given increase in Ca²⁺, whereas low-frequency oscillations activated only NF-κB.

Ca²⁺-Signaling Microdomains

Recent studies by Santana and coworkers suggest a model in which the scaffolding protein AKAP250 targets PKC and calcineurin to caveolin-containing membrane microdomains, where they associated with VDCCs to form a signaling unit capable of mediating persistent Ca²⁺ sparklets (Santana and Navedo 2009). These studies further indicate that persistent VDCC-mediated Ca²⁺ sparklets activate the Ca²⁺-calmodulin-dependent transcription factor NFATc3 (Nieves-Cintron et al. 2008), which modulates expression of the Kv2.1 voltage-dependent K⁺ channel and the β1 subunit of the BK_Ca channel in these cells (Amberg et al. 2004). By extension, similar complexes of P2X₁Rs with kinases and phosphatases might form Ca²⁺-signaling microdomains in postjunctional smooth muscle cell membranes, enabling nerve-evoked purinergic transients to regulate activation of NFAT or other Ca²⁺-sensitive transcription factors.

CONCLUSIONS

In addition to global elevations in intracellular Ca²⁺ mediated by VDCCs, smooth muscle shows a variety of local Ca²⁺ signals, including Ca²⁺ sparks (RyRs), Ca²⁺ puffs (IP₃Rs), Ca²⁺ waves (IP₃Rs/RyRs), junctional Ca²⁺ transients (P2X₁Rs), Ca²⁺ flashes (VDCCs), and Ca²⁺ sparklets (VDCCs). Each signal represents the manifestation of different molecular circuits, which collectively serve to modulate membrane potential, contractility, and gene expression.