Excitation-contraction Coupling In Skeletal Muscle Research Articles

Interaction between the dihydropyridine receptor Ca 2+ channel β-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR_ _R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR_ _R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.

The β 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle

Homozygous zebrafish of the mutant relaxed (red(ts25)) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca(2+) transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the alpha(1S) subunit is normal, whereas the beta(1a) subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the beta(1a) protein in mutant muscle. Thus, the immotile zebrafish relaxed is a beta(1a)-null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the alpha(1S) subunit in the absence of beta(1a). Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an approximately 2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the beta(1a) subunit does not prevent triad targeting of the DHPR alpha(1S) subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in beta(1)-null model organisms.

Multiple Loops of the Dihydropyridine Receptor Pore Subunit Are Required for Full-Scale Excitation-Contraction Coupling in Skeletal Muscle

Understanding which cytosolic domains of the dihydropyridine receptor participate in excitation-contraction (EC) coupling is critical to validate current structural models. Here we quantified the contribution to skeletal-type EC coupling of the α1S (Ca V1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of α1S. Chimeras consisting of α1C (Ca V1.2) with α1S substitutions at each of the interrepeat loops (I-II, II-III, and III-IV loops) and N- and C-terminal domains were evaluated in dysgenic ( α1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca 2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the α1C/ α1S II-III loop chimera, but none of the other single-loop chimeras, recovered a sigmoidal fluorescence-voltage curve indicative of skeletal-type EC coupling. To quantify Ca 2+ transients in the absence of inward Ca 2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of α1C. The Ca 2+ transients expressed by the α1C(E736K)/ α1S II-III loop chimera were ∼70% smaller than those expressed by the Ca 2+-conducting α1C/ α1S II-III variant. The low skeletal-type EC coupling expressed by the α1C/ α1S II-III loop chimera was confirmed in the Ca 2+-conducting α1C/ α1S II-III loop variant using Cd 2+ (10 −4 M) as the Ca 2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca 2+ transients expressed by an α1C/ α1S chimera carrying all tested skeletal α1S domains (all α1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type α1S, and did not change in the presence of the E736K mutation or in the presence of 10 −4 M Cd 2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca 2+ permeant α1S(E1014K) variant, the α1C(E736K)/ α1S II-III loop chimera, and the α1C(E736K)/ α1S chimera carrying all tested α1S domains. The data indicate that the functional recovery produced by the α1S II-III loop is incomplete and that multiple cytosolic domains of α1S are necessary for a quantitative recovery of the EC-coupling phenotype of skeletal myotubes. Thus, despite the importance of the II-III loop there may be other critical determinants in α1S that influence the efficiency of EC coupling.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

The Ca2+ Channel α2δ-1 Subunit Determines Ca2+ Current Kinetics in Skeletal Muscle but Not Targeting of α1S or Excitation-Contraction Coupling

Auxiliary channel subunits regulate membrane expression and modulate current properties of voltage-activated Ca(2+) channels and thus are involved in numerous important cell functions, including muscle contraction. Whereas the importance of the alpha(1S), beta(1a), and gamma Ca(2+) channel subunits in skeletal muscle has been determined by using null-mutant mice, the role of the alpha(2)delta-1 subunit in skeletal muscle is still elusive. We addressed this question by small interfering RNA silencing of alpha(2)delta-1 in reconstituted dysgenic (alpha(1S)-null) myotubes and in BC3H1 skeletal muscle cells. Immunofluorescence labeling of the alpha(1S) and alpha(2)delta-1 subunits and whole cell patch clamp recordings demonstrated that triad targeting and functional expression of the skeletal muscle Ca(2+) channel were not compromised by the depletion of the alpha(2)delta-1 subunit. The amplitudes and voltage dependences of L-type Ca(2+) currents and of the depolarization-induced Ca(2+) transients were identical in control and in alpha(2)delta-1-depleted muscle cells. However, alpha(2)delta-1 depletion significantly accelerated the current kinetics, most likely by the conversion of slowly activating into fast activating Ca(2+) channels. Reverse transcription-PCR analysis indicated that alpha(2)delta-1 is the exclusive isoform expressed in differentiated BC3H1 cells and that depletion of alpha(2)delta-1 was not compensated by the up-regulation of any other alpha(2)delta isoform. Thus, in skeletal muscle the Ca(2+) channel alpha(2)delta-1 subunit functions as a major determinant of the characteristic slow L-type Ca(2+) current kinetics. However, this subunit is not essential for targeting of Ca(2+) channels or for their primary physiological role in activating skeletal muscle excitation-contraction coupling.

Metabolic Biotinylation as a Probe of Supramolecular Structure of the Triad Junction in Skeletal Muscle

Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum. However, it remains uncertain what regions, if any, of the two proteins interact with one another. Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions. Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR. To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site. All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling. To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop. However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1). In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1. These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1.

Mapping Sites of Potential Proximity between the Dihydropyridine Receptor and RyR1 in Muscle Using a Cyan Fluorescent Protein-Yellow Fluorescent Protein Tandem as a Fluorescence Resonance Energy Transfer Probe

Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum. In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the alpha1S and beta1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S. Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor.

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle.

The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.

Involvement of a Heptad Repeat in the Carboxyl Terminus of the Dihydropyridine Receptor β1a Subunit in the Mechanism of Excitation-Contraction Coupling in Skeletal Muscle

Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) β1a subunit and the heterologous cardiac/brain β2a subunit were used to determine which regions of β1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in β1 knockout myotubes and then voltage-clamped with simultaneous measurement of confocal fluo-4 fluorescence. All chimeras expressed a similar density of DHPR charge movements, indicating that the membrane density of DHPR voltage sensors was not a confounding factor in these studies. The data indicates that a β1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues ( β1a 478–524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of β1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which β1aD5 was swapped by the D5 region of β2a expressed Ca 2+ transients triggered by the Ca 2+ current, or none at all. A hydrophobic heptad repeat is present in domain D5 of β1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant β1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (Δ F/ F max = 0.4 ± 0.1 vs. 2.7 ± 0.5 for wild-type β1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, β1a(S481A/L488A/S495A), was normal (Δ F/ F max = 2.1 ± 0.4). In summary, the presence of the β1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in β1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of β1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

The role of calcium and calcium/calmodulin-dependent kinases in skeletal muscle plasticity and mitochondrial biogenesis.

Intracellular Ca(2+) plays an important role in skeletal muscle excitation-contraction coupling and also in excitation-transcription coupling. Activity-dependent alterations in muscle gene expression as a result of increased load (i.e. resistance or endurance training) or decreased activity (i.e. immobilization or injury) are tightly linked to the level of muscle excitation. Differential expression of genes in slow- and fast-twitch fibres is also dependent on fibre activation. Both these biological phenomena are, therefore, tightly linked to the amplitude and duration of the Ca(2+) transient, a signal decoded downstream by Ca(2+)-dependent transcriptional pathways. Evidence is mounting that the calcineurin-nuclear factor of activated T-cells pathway and the Ca(2+)/calmodulin-dependent kinases (CaMK) II and IV play important roles in regulating oxidative enzyme expression, mitochondrial biogenesis and expression of fibre-type specific myofibrillar proteins. CaMKII is known to decode frequency-dependent information and is activated during hypertrophic growth and endurance adaptations. Thus, it was hypothesized that CaMKII, and possibly CaMKIV, are down regulated during muscle atrophy and levels of expression of CaMKII alpha, -II beta, -II gamma and -IV were assessed in skeletal muscles from young, aged and denervated rats. The results indicate that CaMKII gamma, but not CaMKIIalpha or -beta, is up regulated in aged and denervated soleus muscle and that CaMKIV is absent in skeletal but not cardiac muscle. Whether CaMKII gamma up-regulation is part of the pathology of wasting or a result of some adaptational response to atrophy is not known. Future studies will be important in determining whether insights from the adaptational response of muscle to increased loads will provide pharmacological approaches for increasing muscle strength or endurance to counter muscle wasting.

Dantrolene – A review of its pharmacology, therapeutic use and new developments

Human malignant hyperthermia is a life-threatening genetic sensitivity of skeletal muscles to volatile anaesthetics and depolarizing neuromuscular blocking drugs occurring during or after anaesthesia. The skeletal muscle relaxant dantrolene is the only currently available drug for specific and effective therapy of this syndrome in man. After its introduction, the mortality of malignant hyperthermia decreased from 80% in the 1960s to < 10% today. It was soon discovered that dantrolene depresses the intrinsic mechanisms of excitation-contraction coupling in skeletal muscle. However, its precise mechanism of action and its molecular targets are still incompletely known. Recent studies have identified the ryanodine receptor as a dantrolene-binding site. A direct or indirect inhibition of the ryanodine receptor, the major calcium release channel of the skeletal muscle sarcoplasmic reticulum, is thought to be fundamental in the molecular action of dantrolene in decreasing intracellular calcium concentration. Dantrolene is not only used for the treatment of malignant hyperthermia, but also in the management of neuroleptic malignant syndrome, spasticity and Ecstasy intoxication. The main disadvantage of dantrolene is its poor water solubility, and hence difficulties are experienced in rapidly preparing intravenous solutions in emergency situations. Due to economic considerations, no other similar drugs have been introduced into routine clinical practice.

Structural Requirements of the Dihydropyridine Receptor α1S II-III Loop for Skeletal-type Excitation-Contraction Coupling

Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.

Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.

Activating the ryanodine receptor with dihydropyridine receptor II-III loop segments: size and charge do matter.

Excitation-contraction coupling in skeletal muscle is thought to depend on a physical interaction between II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor (DHPR) and ryanodine receptor (RyR). A peptide corresponding to II-III loop residues 671-690 of the skeletal DHPR (peptide A) is a high affinity activator of the RyR when it adopts an alpha-helical structure with critical basic residues aligned along one helical surface (1). Neither the structure of the full length II-III loop, or of sequences longer than 671-690 residues have been determined. Here we describe the structure and function of a 40 amino acid peptide corresponding to residues 671-710 (peptide AB) of the skeletal DHPR alpha1 subunit. This peptide contains the A region with a further 20 residues towards the C-terminus of the II-III loop. We predicted that peptide AB would strongly activate the RyR, because (a) it contains the active A sequence of basic residues and (b) it contains a greater proportion of the II-III loop. The structure of the AB peptide was determined and it was found to consist of two helical regions joined by an unstructured linker region. Surprisingly, although the structure of the A region was maintained, the 40 residue peptide was unable to release Ca2+ from skeletal SR. Strong activity was restored when four negatively charged residues in the C-terminal part of the peptide were replaced by neutral residues. The charge substitution caused minimal changes in the overall structural profile of the peptide and virtually no changes in the A portion of the peptide. The results suggest that the ability of the A region of the skeletal II-III loop to interact with the RyR could depend on the tertiary conformation of the II-III loop, which is thought to change during EC coupling.

The Novel Skeletal Muscle Sarcoplasmic Reticulum JP-45 Protein: MOLECULAR CLONING, TISSUE DISTRIBUTION, DEVELOPMENTAL EXPRESSION, AND INTERACTION WITH α1.1 SUBUNIT OF THE VOLTAGE-GATED CALCIUM CHANNEL

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.

The L-type calcium channel C-terminus: sparking interest beyond its role in calcium-dependent inactivation

It is well established that members of the Cav1 (L-type) calcium channel family trigger a number of critical cellular responses. In skeletal muscle, Cav1.1 calcium channels mediate excitation-contraction (EC) coupling by acting as voltage sensors that trigger the opening of ryanodine receptors and, thus, calcium release from the sarcoplasmic reticulum (SR). The changes in membrane potential are transduced to the ryanodine receptor through physical interactions with the second intracellular loop of the Cav1.1 calcium channel α1 subunit (Tanabe et al. 1990). This interaction also alters L-type calcium channel function, thereby providing for a possible feedback mechanism for the regulation of skeletal muscle EC coupling (Nakai et al. 1996). In heart and smooth muscle, calcium influx through Cav1.2 channels results in calcium-induced calcium release from ryanodine-sensitive internal stores, which subsequently triggers muscle contraction. This process is initiated by calcium ‘sparks’ (i.e. highly localized rises in intracellular calcium concentrations near the inner mouth of the L-type calcium channel pore; Cheng et al. 1993). Similar to the feedback regulation of skeletal muscle EC coupling, the increase in local calcium concentration regulates calcium channel activity per se through calcium-dependent inactivation, a process that is critically dependent on calmodulin binding to an IQ motif in the C-terminal region of the L-type calcium channel α1 subunit (Peterson et al. 1999). The introduction of dominant negative calmodulin mutants into cardiac myocytes disrupts calcium-dependent inactivation, which then culminates in a fivefold prolongation of cardiac action potentials (Alseikhan et al. 2002). This clearly underlines the physiological significance of such feedback inhibition. An article by Woo et al. (2003) in this issue of The Journal of Physiology suggests an additional function of the L-type channel C-terminus-calmodulin complex in regulating calcium sparks and calcium-induced calcium release in cardiac myocytes. Woo and colleagues used a combination of electrophysiological recordings and two-dimensional confocal calcium imaging to explore the role of the Cav1.2 C-terminus in the regulation of calcium signalling in cardiac myocytes. Intracellular dialysis of myocytes with a short Cav1.2 C-terminus calmodulin-binding peptide (located upstream of the IQ locus) resulted in an increase in the frequency of spontaneous calcium sparks and in an increased rate of caffeine-induced SR calcium release. Although L-type calcium channel function per se was not affected, the overall size of the calcium transients was increased. Mutant peptides that are incapable of associating with calmodulin were ineffective, indicating that the regulation of calcium sparks is dependent on a physical association between the L-type calcium channel C-terminus and calmodulin. Surprisingly, the observed effects occurred selectively at the central portion of the myocyte where, unlike at the periphery, calcium channels and ryanodine receptors do not appear to be colocalized. The latter observation suggests that the Cav1.2-C-terminus-calmodulin complex only has access to those ryanodine receptors that are not already colocalized with calcium channels, thus perhaps implying that the receptors expressed at the periphery are already sensitized by interactions with endogenous L-type channels. Taken together, the authors present intriguing evidence suggesting that cardiac ryanodine receptor function, and thus presumably cardiac muscle contraction, could be regulated by dynamic interactions with the L-type calcium channel-calmodulin complex. In view of evidence showing that calmodulin interactions with the C-terminus of Cav1.2 calcium channels triggers cAMP response element-binding protein (CREB)-mediated gene transcription in neurons (Dolmetsch et al. 2001; Mermelstein et al. 2001), this suggests that the calmodulin-L-type channel complex may act as a multifaceted signalling entity in excitable cells. Although the data presented by Woo et al. are interesting, further experimentation will be necessary to confirm the presence of L-type channel-calmodulin-ryanodine receptor complexes in cardiomyocytes and to elucidate the exact underlying molecular determinants, such as the identification of interaction sites on the ryanodine receptor molecule. Moreover, it will be necessary to establish a putative role of these interactions in regulating cardiac action potential properties, perhaps by utilizing elegant viral infection approaches such as those developed by the Yue laboratory (Aklseikhan et al. 2002). Finally, in view of increasing evidence of a voltage-dependent component of cardiac EC coupling (for review, see Ferrier & Howlett, 2001), it will be important to examine whether voltage-dependent changes in the interactions between calmodulin, L-type channels and cardiac ryanodine receptors could account for such a mechanism. Clearly, the regulation of calcium signalling in cardiac myocytes remains a complex issue. Nonetheless, the work of Woo et al. has opened novel avenues for investigations that may ultimately bring us closer to understanding the molecular details underlying the control of cardiac function.

Biomimetic model of skeletal muscle isometric contraction: II. A phenomenological model of the skeletal muscle excitation–contraction coupling process

This paper describes a new macroscopic, phenomenological model of the skeletal muscle excitation–contraction coupling process, as represented by four principal and consecutive compartments (biophysical, biochemical, and biomechanical phases) characteristic of isometric excitation–contraction coupling in mammalian skeletal muscle, and coupled by a system of simultaneous, first-order linear ordinary differential equations. The model is based upon biological compartmental transport kinetics and irreversible thermodynamic energy transformation, and represents a distinct improvement over other biomimetic models. The model was derived using physiological parameter data published in the literature, and validated using MATLAB R 12 TM .

The C Terminus (Amino Acids 75–94) and the Linker Region (Amino Acids 42–54) of the Ca2+-binding Protein S100A1 Differentially Enhance Sarcoplasmic Ca2+ Release in Murine Skinned Skeletal Muscle Fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.

Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle fibers and cytosolic [Ca2+] in myotubes

In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle fibers of the rat. The nonspecific PLA2 inhibitor indomethacin (200 microM) significantly increased the peak (approximately 2-fold, P = 0.02) and the width (approximately 6-fold, P = 0.008) of depolarization-induced force responses (DIFRs) elicited in the fibers (n = 4). Exposure of the skinned EDL fibers to indomethacin (200 microM) (n = 7) and another PLA2 inhibitor quinacrine (200 microM) (n = 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 microM), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myofibrillar [Ca2+]. Indomethacin (200 microM) produced a small but significant increase in the Ca2+ sensitivity of the contractile apparatus of skinned EDL fibers and the maximum force production. Indomethacin (200 microM) also had significant effects on SR function, increasing SR Ca2+ loading in the skinned fibers (117.2 +/- 3.0% of controls, P = 0.0008, n = 8) and inducing intracellular Ca2+ release in isolated intact flexor digitorum brevis (FDB) fibers (n = 7) and C2C12 myotubes (n = 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle.