The mechanisms that link the primary increase in SR Ca²⁺ leak of MH susceptibility and related conditions to their disease phenotypes are not well understood. We found that abnormal Ca²⁺ homeostasis in MHS individuals induces proteolysis of junctophilin1 (JPh1), an essential structural protein of EC coupling (Perni, in 2017). Guo (in 2018) and Lahiri (in 2020) reported similar fragmentation of JPh2 in stressed hearts. Western blot of patients’ muscle with domain-specific antibodies showed a deficit of full-length JPh1 and excess of a 44-kD C-terminal fragment (JPh44) in MHS subjects. While JPh1 was located in T-SR junctions, JPh44 was found anywhere within the I band, and at high densities within nuclei—a location forbidden for JPh1. Expression and cleavage in mice of a JPh1 plasmid tagged at both ends showed that its N-terminal fragment remained in triads, and the C-terminal fragment, orthologue to JPh44, entered nuclei, which indicates that JPh44 is the C-terminal cleavage product. Endogenous calpain1 appeared in T-SR junctions, colocalized with JPh1. On muscle extracts and primary cultures, Ca²⁺-activated calpain1 cleaved a 44-kD JPh1 piece, consistent with the C-terminal fragment that starts at Ser241, the highest probability cleavage site found by calpain1 algorithms. Completing the identification of Ser241 as the likely start of JPh44, the tagged deletion plasmid GFP-JPh1_Δ1-240, expressed in mice, copied the location and migration of JPh44. Expression of GFP-JPh1_Δ1-240 in C2C12 myoblasts reduced by more than twofold the transcription of PI3K-Akt genes that inhibit muscle uptake and storage of glucose, including GSK3β, an inhibitor of glycogen synthase that is activated in MHS patients. In agreement with the genetic profile, GSK3β protein content decreased upon expression of GFP-JPh1_Δ1-240. In sum, the identified gene control roles of JPh44 oppose the deleterious effects of chronically elevated cytosolic [Ca²⁺], including late-onset hyperglycemia and type-2 diabetes (Tammineni, in 2020).

Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca²⁺ (Ca_V1.1) channels, found in tubules or plasmalemma, form symmetric complexes called Ca_V tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, Ca_V tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor–lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between Ca_Vs and RYRs and test this hypothesis using a quantitative model of the energies that govern Ca_V1.1–RYR1 binding, which are assumed to depend on number and location of bound Ca_Vs. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by Ca_Vs, so that Ca_Vs positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two Ca_Vs bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.

Ryanodine-sensitive intracellular Ca²⁺ channels (RyRs) open upon binding Ca²⁺ at cytosolic-facing sites. This results in concerted, self-reinforcing opening of RyRs clustered in specialized regions on the membranes of Ca²⁺ storage organelles (endoplasmic reticulum and sarcoplasmic reticulum), a process that produces Ca²⁺-induced Ca²⁺ release (CICR). The process is optimized to achieve large but brief and localized increases in cytosolic Ca²⁺ concentration, a feature now believed to be critical for encoding the multiplicity of signals conveyed by this ion. In this paper, I trace the path of research that led to a consensus on the physiological significance of CICR in skeletal muscle, beginning with its discovery. I focus on the approaches that were developed to quantify the contribution of CICR to the Ca²⁺ increase that results in contraction, as opposed to the flux activated directly by membrane depolarization (depolarization-induced Ca²⁺ release [DICR]). Although the emerging consensus is that CICR plays an important role alongside DICR in most taxa, its contribution in most mammalian muscles appears to be limited to embryogenesis. Finally, I survey the relevance of CICR, confirmed or plausible, to pathogenesis as well as the multiple questions about activation of release channels that remain unanswered after 50 years.

In skeletal muscle, the four-helix voltage-sensing modules (VSMs) of Ca_V1.1 calcium channels simultaneously gate two Ca²⁺ pathways: the Ca_V1.1 pore itself and the RyR1 calcium release channel in the sarcoplasmic reticulum. Here, to gain insight into the mechanism by which VSMs gate RyR1, we quantify intramembrane charge movement associated with VSM activation (sensing current) and gated Ca²⁺ release flux in single muscle cells of mice and rats. As found for most four-helix VSMs, upon sustained depolarization, rodent VSMs lose the ability to activate Ca²⁺ release channels opening; their properties change from a functionally capable mode, in which the mobile sensor charge is called charge 1, to an inactivated mode, charge 2, with a voltage dependence shifted toward more negative voltages. We find that charge 2 is promoted and Ca²⁺ release inactivated when resting, well-polarized muscle cells are exposed to low extracellular [Ca²⁺] and that the opposite occurs in high [Ca²⁺]. It follows that murine VSMs are partly inactivated at rest, which establishes the reduced availability of voltage sensing as a pathogenic mechanism in disorders of calcemia. We additionally find that the degree of resting inactivation is significantly different in two mouse strains, which underscores the variability of voltage sensor properties and their vulnerability to environmental conditions. Our studies reveal that the resting and activated states of VSMs are equally favored by extracellular Ca²⁺. Promotion by an extracellular species of two states of the VSM that differ in the conformation of the activation gate requires the existence of a second gate, inactivation, topologically extracellular and therefore accessible from outside regardless of the activation state.

A novel category of diseases of striated muscle is proposed, the couplonopathies, as those that affect components of the couplon and thereby alter its operation. Couplons are the functional units of intracellular calcium release in excitation–contraction coupling. They comprise dihydropyridine receptors, ryanodine receptors (Ca²⁺ release channels), and a growing list of ancillary proteins whose alteration may lead to disease. Within a generally similar plan, the couplons of skeletal and cardiac muscle show, in a few places, marked structural divergence associated with critical differences in the mechanisms whereby they fulfill their signaling role. Most important among these are the presence of a mechanical or allosteric communication between voltage sensors and Ca²⁺ release channels, exclusive to the skeletal couplon, and the smaller capacity of the Ca stores in cardiac muscle, which results in greater swings of store concentration during physiological function. Consideration of these structural and functional differences affords insights into the pathogenesis of several couplonopathies. The exclusive mechanical connection of the skeletal couplon explains differences in pathogenesis between malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT), conditions most commonly caused by mutations in homologous regions of the skeletal and cardiac Ca²⁺ release channels. Based on mechanistic considerations applicable to both couplons, we identify the plasmalemma as a site of secondary modifications, typically an increase in store-operated calcium entry, that are relevant in MH pathogenesis. Similar considerations help explain the different consequences that mutations in triadin and calsequestrin have in these two tissues. As more information is gathered on the composition of cardiac and skeletal couplons, this comparative and mechanistic approach to couplonopathies should be useful to understand pathogenesis, clarify diagnosis, and propose tissue-specific drug development.

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

Imaging, optical mapping, and optical multisite recording of transmembrane potential (V_m) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V_m. Accordingly, they have been used for monitoring V_m in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER—the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands—was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast “slit scanner” increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.

Parameters (amplitude, width, kinetics) of Ca²⁺ sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x–y images in three planes at 1-µm separation (x-y-z-t mode). In 3,000 x–y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F₀ = 0.7. The Ca²⁺ release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca²⁺ release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca²⁺ in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca²⁺]_SR) or that different channel clusters respond to [Ca²⁺]_SR with different sensitivity.

The mechanisms that terminate Ca²⁺ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca²⁺ concentration inside the sarcoplasmic reticulum (SR), [Ca²⁺]_SR, simultaneously with that in the cytosol, [Ca²⁺]_c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca²⁺ release flux (derived from [Ca²⁺]_c(t)) over the gradient that drives it (essentially equal to [Ca²⁺]_SR) provided directly, for the first time, a dynamic measure of the permeability to Ca²⁺ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca²⁺]_SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca²⁺ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca²⁺]_SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca²⁺ release.

Current fluorescent monitors of free [Ca²⁺] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca²⁺. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective K_D of 222 µM and a dynamic range [(R_max − R_min)/R_min] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, R_max, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca²⁺]_SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca²⁺]_SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca²⁺ concentration.

Contractile activation in striated muscles requires a Ca²⁺ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca²⁺] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca²⁺ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca²⁺ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca²⁺ permeability and inversely to its Ca²⁺ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca²⁺ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca²⁺ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca²⁺], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca²⁺, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca²⁺ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca²⁺ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.

An algorithm for the calculation of Ca²⁺ release flux underlying Ca²⁺ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca²⁺ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca²⁺ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca²⁺ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca²⁺ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation–contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca²⁺ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation–contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca²⁺ currents were monitored when 2–30 mM Ca²⁺ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca²⁺ current (at 0 mV holding potential) and lumenal [Ca²⁺] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH₃SO₃ concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca²⁺; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca²⁺ (n > 6). Unitary Ca²⁺ current was also defined in the presence of symmetrical [Mg²⁺] (1 mM) and low [Cs⁺] (5 mM). Under these conditions, unitary Ca²⁺ current in 2 and 10 mM lumenal Ca²⁺ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs⁺] (50 mM), Mg²⁺ (1 mM), and lumenal [Ca²⁺] (10 mM), unitary Ca²⁺ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs⁺ and Mg²⁺ on unitary Ca²⁺ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg²⁺ effectively compete with Ca²⁺ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca²⁺ is 2 mM, then our results indicate that unitary Ca²⁺ current under physiological conditions should be <0.6 pA.

In studies of gating currents of rabbit cardiac Ca channels expressed as α_1C/β_2a or α_1C/β_2a/α₂δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V_1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V_1/2 ≃ −90 mV) is generated in inactivated channels. In α_1C/β_2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α₂δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α₂δ subunit did not change the V_1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α₂δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

This is a quantitative model of control of Ca²⁺ release from the sarcoplasmic reticulum in skeletal muscle, based on dual control of release channels (ryanodine receptors), primarily by voltage, secondarily by Ca²⁺ (Ríos, E., and G. Pizarro. 1988. NIPS. 3:223–227). Channels are positioned in a double row array of between 10 and 60 channels, where exactly half face voltage sensors (dihydropyridine receptors) in the transverse (t) tubule membrane (Block, B.A., T. Imagawa, K.P. Campbell, and C. Franzini-Armstrong. 1988. J. Cell Biol. 107:2587–2600). We calculate the flux of Ca²⁺ release upon different patterns of pulsed t-tubule depolarization by explicit stochastic simulation of the states of all channels in the array. Channels are initially opened by voltage sensors, according to an allosteric prescription (Ríos, E., M. Karhanek, J. Ma, A. González. 1993. J. Gen. Physiol. 102:449–482). Ca²⁺ permeating the open channels, diffusing in the junctional gap space, and interacting with fixed and mobile buffers produces defined and changing distributions of Ca²⁺ concentration. These concentrations interact with activating and inactivating channel sites to determine the propagation of activation and inactivation within the array. The model satisfactorily simulates several whole-cell observations, including kinetics and voltage dependence of release flux, the “paradox of control,” whereby Ca²⁺-activated release remains under voltage control, and, most surprisingly, the “quantal” aspects of activation and inactivation (Pizarro, G., N. Shirokova, A. Tsugorka, and E. Ríos. 1997. J. Physiol. 501:289–303). Additionally, the model produces discrete events of activation that resemble Ca²⁺ sparks (Cheng, H., M.B. Cannell, and W.J. Lederer. 1993. Science (Wash. DC). 262:740–744). All these properties result from the intersection of stochastic channel properties, control by local Ca²⁺, and, most importantly, the one dimensional geometry of the array and its mesoscopic scale. Our calculations support the concept that the release channels associated with one face of one junctional t-tubule segment, with its voltage sensor, constitute a functional unit, termed the “couplon.” This unit is fundamental: the whole cell behavior can be synthesized as that of a set of couplons, rather than a set of independent channels.