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The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked [Ca.sup.2+] transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that [Ca.sup.2+] release was suppressed at all test membrane potentials in [S100A1.sup.-/-] fibers. Here we examine the role of S 100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic [Ca.sup.2+] binding and transport processes to calculate sarcoplasmic reticulum [Ca.sup.2+] release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in [S100A1.sup.-/-] muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced [Ca.sup.2+] release flux, resulting in impaired contractile activation and force production in skeletal muscle. S100; excitation-contraction coupling; calcium signaling; muscle 10.1152/ajpcell.00180.2010.

[Ca.sup.2+] channels play crucial roles in cellular signal transduction and are important targets of pharmacological agents. They are also associated with auxiliary subunits exhibiting functions that are still incompletely resolved. Skeletal muscle L-type [Ca.sup.2+] channels (dihydropyridine receptors, DHPRs) are specialized for the remote voltage control of type 1 ryanodine receptors (RyR1) to release stored [Ca.sup.2+]. The skeletal muscle-specific 1, subunit of the DHPR ([[gamma].sub.1]) down-modulates availability by altering its steady state voltage dependence. The effect resembles the action of certain [Ca.sup.2+] antagonistic drugs that are thought to stabilize inactivated states of the DHPR. In the present study we investigated the cross influence of [[gamma].sub.1] and [Ca.sup.2+] antagonists by using wild-type ([gamma]+/+) and [[gamma].sub.1] knockout ([gamma]-/-) mice. We studied voltage-dependent gating of both L-type [Ca.sup.2+] current and [Ca.sup.2+] release and the allosteric modulation of drug binding. We found that 10 [micro]M diltiazem, a benzothiazepine drug, more than compensated for the reduction in high-affinity binding of the dihydropyridine agent isradipine caused by [[gamma].sub.1] elimination; 5 [micro]M devapamil [(-)D888], a phenylalkylamine [Ca.sup.2+] antagonist, approximately reversed the right-shifted voltage dependence of availability and the accelerated recovery kinetics of [Ca.sup.2+] current and [Ca.sup.2+] release. Moreover, the presence of [[gamma].sub.1] altered the effect of D888 on availability and strongly enhanced its impact on recovery kinetics demonstrating that [[gamma].sub.1] and the drug do not act independently of each other. We propose that the [[gamma].sub.1] subunit of the DHPR functions as an endogenous [Ca.sup.2+] antagonist whose task may be to minimize [Ca.sup.2+] entry and [Ca.sup.2+] release under stress-induced conditions favoring plasmalemma depolarization.

Functional impacts of the skeletal muscle-specific [Ca.sup.2+] channel subunit [[gamma].sub.1] have previously been studied using coexpression with the cardiac [[alpha].sub.1C] polypeptide in nonmuscle cells and primary-cultured myotubes of [[gamma].sub.1]-deficient mice. Data from single adult muscle fibers of [gamma]-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from [gamma]+/+ and [gamma]-/- mice. We measured L-type [Ca.sup.2+] inward currents and intracellular [Ca.sup.2+] transients during 100-ms step depolarizations from a holding potential of -80 mV. Radometric [Ca.sup.2+] transients were analyzed with a removal model fit approach to calculate the flux of [Ca.sup.2+] from the sarcoplasmic reticulum. [Ca.sup.2+] current density [Ca.sup.2+] release flux, and the voltage dependence of activation of both [Ca.sup.2+] current and [Ca.sup.2+] release were not significantly different. By varying the holding potential and recording [Ca.sup.2+] current and [Ca.sup.2+] release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the [Ca.sup.2+] current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of [Ca.sup.2+] release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively; Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in [gamma]-/- compared with [gamma]+/+. This difference was independent of the presence of extracellular [Ca.sup.2+] and likely results from the lower sensitivity to voltage-dependent inactivation of [Ca.sup.2+] release. These results demonstrate a specific alteration of voltage-dependent inactivation of both [Ca.sup.2+] entry and [Ca.sup.2+] release by the [[gamma].sub.1] submit of the dihydropyridine receptor in mature muscle fiber's of file moose. KEY WORDS: mammalian skeletal muscle * excitation--contraction coupling * accessory subunits * knockout mouse * voltage

Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca2+-dependent functions is contraction of smooth and striated muscle tissue, which results from Ca2+ ligation to calmodulin and troponin C, respectively. Ca2+ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca2+-dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca2+ channels. Ca2+ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca2+ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO), which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1. [ABSTRACT FROM AUTHOR]

Sparks are transient local elevations of Ca ion concentration observed in different types of muscle cells. Such local [Ca.sup.2+] signals can be provoked in skeletal muscle cells by altering the osmotic pressure of the extracellular solution. In this issue, Weisleder et al. (see p. 639) demonstrate that the [Ca.sup.2+] response to osmotic stress is substantially altered in aged muscle. The study presents evidence for a link between this finding and a reduced expression of mitsugumin 29 (MG29), a small membrane protein of the sarcoplasmic reticulum (SR).

Skeletal muscle excitation--contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and L-type Ca2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca2+ channel is still enigmatic. Here we show that Ca2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca2+ influx is eliminated. Homozygous knock-in mice display SR Ca2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca2+ influx in mammalian skeletal muscle is an evolutionary remnant. [ABSTRACT FROM AUTHOR]

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca2+ concentrations, whereas at micromolar Ca2+ concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1D/D) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1D/D mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1D/D mice had depressed Ca2+ transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca2+ transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1D/D mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1D/D fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca2+ release flux, consistent with increased summation of the Ca2+ transient and contractile force. Peak Ca2+ release flux was suppressed at all voltages in voltage-clamped Ryr1D/D fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca2+ release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

Ca2+ channels play crucial roles in cellular signal transduction and are important targets of pharmacological agents. They are also associated with auxiliary subunits exhibiting functions that are still incompletely resolved. Skeletal muscle L-type Ca2+ channels (di-hydropyridine receptors, DHPR5) are specialized for the remote voltage control of type 1 ryanodine receptors (RyR1) to release stored Ca2+. The skeletal muscle-specific γ subunit of the DHPR (γ1) down-modulates availability by altering its steady state voltage dependence. The effect resembles the action of certain Ca2+ antagonistic drugs that are thought to stabilize inactivated states of the DHPR. In the present study we investigated the cross influence of γ1 and Ca2+ antagonists by using wild-type (γ+/+) and γ1 knockout (γ-/-) mice. We studied voltage-dependent gating of both L-type Ca2+ current and Ca2+ release and the allosteric modulation of drug binding. We found that 10 μM diltiazem, a benzothiazepine drug, more than compensated for the reduction in high-affinity binding of the dihydropyridine agent isradipine caused by 71 elimination; 5 μM devapamil [(-)D8881. a phenylalkylamine Ca2+ antagonist, approximately reversed the right-shifted voltage dependence of availability and the accelerated recovery kinetics of Ca2+ current and Ca2+ release. Moreover, the presence of γ1 altered the effect of D888 on availability and strongly enhanced its impact on recovery kinetics demonstrating that γ1 and the drug do not act independently of each other. We propose that the γ1 subunit of the DHPR functions as an endoge- nous Ca2+ antagonist whose task may be to minimize Ca2+ entry and Ca2+ release under stress-induced conditions favoring plasmalemma depolarization. [ABSTRACT FROM AUTHOR]