The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.

Ca 2؉ signals, produced by Ca 2؉ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca 2؉ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca 2؉ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca 2؉ release channels have closed. This finding indicates that Ca 2؉ is released from a ''proximate'' compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca 2؉ , which was recorded upon stimulation of prolonged, cell-wide Ca 2؉ release. An intrastore surge upon induction of Ca 2؉ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca 2؉ -adsorbing linear polymers of calsequestrin, they could deliver Ca 2؉ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca 2؉ ] and sustain the concentration gradient that drives release flux.calcium signaling ͉ calcium sparks ͉ excitation-contraction coupling ͉ sarcoplasmic reticulum ͉ skeletal muscle R apid changes in intracellular cytosolic [Ca 2ϩ ] are required for signaling functions in many cell types (1). These changes are achieved via Ca 2ϩ release through channels, ryanodine receptors (RyRs), which must open and close quickly. To increase its speed, gating of RyRs relies on effects of the permeant ion, including channel opening by elevated cytosolic [Ca 2ϩ ] (2). In muscle, the desirable fast kinetic features are already present in its elementary signaling events, Ca 2ϩ sparks (3), which involve the nearly simultaneous opening (4) of a number of channels (5), followed by their synchronized closing (4). Thus, this gating does not follow the usual Markovian rules for channels that evolve independently but requires timekeeping and synchronization (6). In cardiac muscle, depletion of sarcoplasmic reticulum (SR) Ca 2ϩ is the likely timer of channel closing, and the substantial depletion that follows the cardiac beat (7) was imaged as ''blinks'' associated with Ca 2ϩ sparks (8). The sensor that translates depletion into channel closing appears to be the main intra-SR buffer, calsequestrin (CSQ) (9).By contrast, in skeletal muscle, depletion associated with a twitch is only 8-15% (10). This low rate of depletion reflects a SR with larger terminal cisternae containing higher concentrations of a CSQ of greater binding capacity, thus constituting a much greater calcium reservoir. Despite the greater store, sparks of skeletal mus...

beta-Adrenergic stimulation of the heart is thought to increase cardiac muscle contractility by activation of cyclic AMP-dependent protein kinase and concomitant increase in the phosphorylation of certain proteins (for refs see refs 1-6). Electrophysiological studies have shown that the stimulation of cardiac beta-adrenoreceptors, the external application of cyclic AMP or its analogues to Purkinje fibres, or the injection of cyclic AMP into single myocytes can increase the slow inward current (Isi) during the plateau phase of the action potential (AP). In heart muscle this current is mainly carried by Ca2+ (refs 10, 11) and it has been suggested that cyclic AMP-dependent phosphorylation of some component of the calcium channel increases the amount of Ca2+ which enters the cell during depolarization. We have investigated this hypothesis by examining the electrical responses of isolated guinea pig ventricular myocytes to pressure injections of subunits of the cyclic AMP-dependent protein kinase. We report here that injection of the catalytic subunit (C) resulted in a lengthening of the action potential duration (APD) and an increase in the height of the plateau as well as the amplitude of Isi. By contrast, the injection of regulatory subunit (R) shortened the APD of fast and slow response APs, an effect which was reversed by adrenaline.

SUMMARY1. The effect of low extracellular free calcium ion concentration ([Ca2+]

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were “lone embers.” Average spatial width was ∼2 μm in the rat and 1.5 μm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37–160 channels opened in the spark. In simulations, 25–60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 μm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35–260 channels. Not infrequently, a few of those remain open and produce the trailing ember.