Calmodulin Regulation Research Articles

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.

Calmodulin regulation of CaV channels is a prominent Ca2+ feedback mechanism orchestrating vital adjustments of Ca2+ entry. The long-held structural correlate of this regulation has been Ca2+-bound calmodulin complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (iTL analysis). Accordingly, we uncover frank exchange of Ca2+-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca2+-calmodulin binds an NSCaTE module on the channel amino terminus, while the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.

Impaired intracellular Ca2+ signaling alters the activity of growth promoting signaling molecules in Ryr2ADA/ADA mice

Substitution of three amino acid residues in the calmodulin (CaM) binding domain of the cardiac ryanodine receptor ion channel (W3587A, L3591D, F3603A, RyR2ADA) impairs CaM inhibition of RyR2, alters intracellular Ca2+ handling and leads to rapid increase in heart size and death of Ryr2ADA/ADA mice within about 2 weeks of birth (Yamaguchi et al., J Clin Invest 117: 1344–1353, 2007). To elucidate mechanisms by which cardiac heart size is increased, we determined the protein and phosphorylation levels of signaling molecules in hearts of 1‐day and 10‐day old Ryr2ADA/ADA and wild type mice. Immunoblots showed a significant increase in phosphorylation levels of mTOR at Ser2448, p70 S6 kinase at Thr389, S6 ribosomal protein at Ser235/236 and 240/244 and eukaryotic translation initiation factor 4B (eIF4B) at Ser422 with no major changes in protein expression levels. Phosphorylation of AMP‐activated protein kinase (AMPK) at Thr172 was decreased, whereas phosphorylation of insulin receptor substrate 1 (IRS1) at Ser612, p38 mitogen‐activated protein kinase (p38MAPK) at Thr180/Tyr182, phosphoinositide kinase 1 (PDK1) at Ser241 and p90 ribosomal S6 kinases (p90RSKs) at Ser380 and Thr 359/Ser 363 were all increased at postnatal day 10 in Ryr2ADA/ADA mice. The results indicate that impaired CaM regulation of RyR2 alters the activity of multiple signaling pathways regulating cardiac metabolism and growth. Supported by NIH.

How CaBPs Prevail over CaM in Modulating Cav1 Channels

Calcium-binding proteins (CaBPs), a subfamily of calmodulin (CaM)-like proteins, are widely expressed throughout the brain. They are believed to compete with CaM for binding to target molecules, thereby exerting vital modifications of Ca2+/CaM regulation. Yet, deep sequencing of brain reveals CaBP transcript levels 100-10000 times lower than for CaM. How could so little CaBP produce effects in the brain? Here, we examine this fundamental paradox in L-type CaV1.3 Ca2+ channels, whose Ca2+/CaM-dependent inactivation (CDI) can be suppressed by CaBP4. Upon fusion of CaM to CaV1.3 (a), the ultra-high local CaM concentration should occlude CaBP4 access to a common site. Remarkably, separately expressed CaBP4 still eliminates CDI (b), arguing for different CaBP4 and CaM sites. FRET assays map these distinct sites, and establish corresponding association constants for binding. This noncompetitive scheme predicts that micromolar CaBP4 should reduce CDI, even with abundant CaM. To test this, we used patch fluorometry to measure CDI while gauging the concentration of fluorescently labeled CaBP4 (c). CaBP4 indeed halves CDI by ∼0.5 uM (d, measured), with abundant CaM throughout. Hence, CaBP molecules may prevail over CaM by utilizing distinct sites on target molecules across the brain.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Regulation of Connexin26 by Ca2+ and Calmodulin

Gap junction channels are key pathways for direct cell-cell communication that enables the intercellular diffusion of ions, second messengers, small metabolites and electrical coupling. To date, there are over 20 members of the gap junction channel family identified in human and they serve distinct roles in different cells and tissues. Among them, Cx26 is one of the most prominently expressed connexins (including Cx26, 29, 30 and 43) in the cochlea, which is primarily responsible of sound transduction in the inner ear. Over 100 mutations of Cx26 genes account for the 14% of the hearing loss. Here, we report a novel calcium binding pocket conserved in various family of Cxs using our established computer algorithms. In addition, we have also identified a calmodulin regulation site that with many disease mutations reside proximately. using a variety of spectroscopic methods including CD, fluorescence, NMR, we have determined metal binding affinity, stoichiometry, conformational change and binding modes of Ca2+ and Calmodulin.

Calcium Concentration Fluctuations and Subspace Volume Influence Calcium-Regulated Calcium Channel Gating and Subspace Dynamics

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough that local signaling may involve 1-100 calcium ions. Therefore, the question arises: is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of subspace calcium concentration and channel open probability (a result that accounts for the small subspace volume and concentration fluctuations) with the corresponding deterministic model (an approximation that assumes large system size and ignores concentration fluctuations). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium-binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast, demonstrating that the calcium concentration fluctuations and subspace volume influence channel gating and subspace dynamics. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers [Tadross, Dick, Yue. Cell 133: 1228-40, 2008]. Additional work will consider the influence of slow and rapid buffers present in the subspace and whether and under what conditions these buffers mitigate the effects of concentration fluctuations on channel gating and subspace dynamics.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor: Ca2+signalling consequences of defective CaM binding to RyR2

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor: Ca2+signalling consequences of defective CaM binding to RyR2

The Neuronal Calcium Sensor Protein Acrocalcin: A Potential Target of Calmodulin Regulation during Development in the Coral Acropora millepora

To understand the calcium-mediated signalling pathways underlying settlement and metamorphosis in the Scleractinian coral Acropora millepora, a predicted protein set derived from larval cDNAs was scanned for the presence of EF-hand domains (Pfam Id: PF00036). This approach led to the identification of a canonical calmodulin (AmCaM) protein and an uncharacterised member of the Neuronal Calcium Sensor (NCS) family of proteins known here as Acrocalcin (AmAC). While AmCaM transcripts were present throughout development, AmAC transcripts were not detected prior to gastrulation, after which relatively constant mRNA levels were detected until metamorphosis and settlement. The AmAC protein contains an internal CaM-binding site and was shown to interact in vitro with AmCaM. These results are consistent with the idea that AmAC is a target of AmCaM in vivo, suggesting that this interaction may regulate calcium-dependent processes during the development of Acropora millepora.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

Protein Gq Modulates Termination of Phototransduction and Prevents Retinal Degeneration

Appropriate termination of the phototransduction cascade is critical for photoreceptors to achieve high temporal resolution and to prevent excessive Ca(2+)-induced cell toxicity. Using a genetic screen to identify defective photoresponse mutants in Drosophila, we isolated and identified a novel Gα(q) mutant allele, which has defects in both activation and deactivation. We revealed that G(q) modulates the termination of the light response and that metarhodopsin/G(q) interaction affects subsequent arrestin-rhodopsin (Arr2-Rh1) binding, which mediates the deactivation of metarhodopsin. We further showed that the Gα(q) mutant undergoes light-dependent retinal degeneration, which is due to the slow accumulation of stable Arr2-Rh1 complexes. Our study revealed the roles of G(q) in mediating photoresponse termination and in preventing retinal degeneration. This pathway may represent a general rapid feedback regulation of G protein-coupled receptor signaling.

FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle

Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 Å. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.

Asynchronous Ca 2 + current conducted by voltage-gated Ca 2+ (Ca V )-2.1 and Ca V 2.2 channels and its implications for asynchronous neurotransmitter release

We have identified an asynchronously activated Ca(2+) current through voltage-gated Ca(2+) (Ca(V))-2.1 and Ca(V)2.2 channels, which conduct P/Q- and N-type Ca(2+) currents that initiate neurotransmitter release. In nonneuronal cells expressing Ca(V)2.1 or Ca(V)2.2 channels and in hippocampal neurons, prolonged Ca(2+) entry activates a Ca(2+) current, I(Async), which is observed on repolarization and decays slowly with a half-time of 150-300 ms. I(Async) is not observed after L-type Ca(2+) currents of similar size conducted by Ca(V)1.2 channels. I(Async) is Ca(2+)-selective, and it is unaffected by changes in Na(+), K(+), Cl(-), or H(+) or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, I(Async) increases in a pulse-wise manner, providing Ca(2+) entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with I(Async). I(Async) is much larger for slowly inactivating Ca(V)2.1 channels containing β(2a)-subunits than for rapidly inactivating channels containing β(1b)-subunits. I(Async) requires global rises in intracellular Ca(2+), because it is blocked when Ca(2+) is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca(2+) binding to calmodulin nor mutations that prevent calmodulin regulation of Ca(V)2.1 block I(Async). The rise of I(Async) during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca(2+), and its enhancement by β(2a)-subunits all resemble asynchronous release, suggesting that I(Async) is a Ca(2+) source for asynchronous neurotransmission.

Discrete-State Stochastic Models of Calcium-Regulated Calcium Influx and Subspace Dynamics Are Not Well-Approximated by ODEs That Neglect Concentration Fluctuations

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough (e.g., 10−17 liters) that local signaling may involve 1–100 calcium ions. Is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of fluctuating subspace calcium concentration (a result that accounts for the small subspace volume) with the corresponding deterministic model (an approximation that assumes large system size). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers.

Molecular Mechanisms of Calmodulin Regulation of TRPV6

Transient Receptor Potential Vanilloid 6 (TRPV6) is an epithelial Ca2+ channel that is responsible for active Ca2+ absorption in the intestines. We have recently demonstrated that this channel is activated directly by phosphatidylinositol 4, 5-bisphosphate (PIP2) and that cytoplasmic ATP stimulates TRPV6 channel activity indirectly by providing substrates for lipid kinases and the generation of PIP2. TRPV6 undergoes Ca2+-induced inactivation, preventing toxic Ca2+ overload of the cell. This inactivation has been shown to be mediated both by Ca2+-calmodulin (CaM) and depletion of PIP2.

The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively

Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.

FRET Detection of Calmodulin Binding to the Cardiac RyR2 Calcium Release Channel

Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.

Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, such as those formed by Sec61 complexes in the ER membrane, would interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism of intracellular signaling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of Sec61α from Canis familiaris that binds CaM, but not Ca2+-free apo-CaM, with nanomolar affinity and sequence specificity. In single channel lipid bilayer measurements, CaM potently mediated Sec61-channel closure in a Ca2+-dependent manner. No functional CaM binding motif was identified in the corresponding region of Sec61p from Saccharomyces cerevisiae, and no channel closure occurred in the presence of CaM and Ca2+. Therefore, CaM binding to the cytosolic N-terminus of Sec61α is involved in limiting Ca2+-leakage from the ER in C. familiaris but not S. cerevisiae.

Dysfunctional ryanodine receptor and cardiac hypertrophy: role of signaling molecules

Mice with three amino acid mutations in the calmodulin binding domain of type-2 ryanodine receptor ion channel (Ryr2(ADA/ADA) mice) have impaired intracellular Ca(2+) handling and cardiac hypertrophy with death at an early age. In this report, the role of signaling molecules implicated in cardiac hypertrophy of Ryr2(ADA/ADA) mice was investigated. Calcineurin A-β (CNA-β) and nuclear factor of activated T cell (NFAT) signaling were monitored in mice carrying either luciferase transgene driven by NFAT-dependent promoter or knockout of CNA-β. NFAT transcriptional activity in Ryr2(ADA/ADA) hearts was not markedly upregulated at embryonic day 16.5 compared with wild-type but significantly increased at postnatal days 1 and 10. Ablation of CNA-β extended the life span of Ryr2(ADA/ADA) mice and enhanced cardiac function without improving sarcoplasmic reticulum Ca(2+) handling or suppressing the expression of genes implicated in cardiac hypertrophy. Embryonic day 16.5 Ryr2(ADA/ADA) mice had normal heart weights with no major changes in Akt1 and class II histone deacetylase phosphorylation and myocyte enhancer factor-2 activity. In contrast, phosphorylation levels of Erk1/2, p90 ribosomal S6 kinases (p90RSKs), and GSK-3β were increased in hearts of embryonic day 16.5 homozygous mutant mice. The results indicate that an impaired calmodulin regulation of RyR2 was neither associated with an altered CNA-β/NFAT, class II histone deacetylase (HDAC)/MEF2, nor Akt signaling in embryonic day 16.5 hearts; rather increased Erk1/2 and p90RSK phosphorylation levels likely leading to reduced GSK-3β activity were found to precede development of cardiac hypertrophy in mice expressing dysfunctional ryanodine receptor ion channel.

Evidence for the possible involvement of calmodulin in regulation of steady state levels of Hsp90 family members (Hsp87 and Hsp85) in response to heat shock in sorghum

Pharmacological studies, using Ca2+ channel blockers (LaCl3 and verapamil) and calmodulin (CaM) antagonists (CPZ and W7), were carried out to understand the role of Ca2+/CaM in the regulation of heat shock-induced expression of Hsp90 (Hsp87 and Hsp85) and Hsp70 (Hsp75 and Hsp73) members in sorghum. It was observed that the expression of both Hsp87 and Hsp85 proteins was decreased in presence of Ca2+ channel blockers and CaM antagonists, under both control and heat stress conditions, as contrary to the steady state levels of Hsp75 and Hsp73, which were not affected significantly under similar conditions. Further, the exposure of sorghum seedlings to geldanamycin, a specific inhibitor of Hsp90, resulted in induction of Hsp87 and Hsp85 in the absence of heat shock also. This study provides the first evidence suggesting that in plants, the in vivo expression of Hsp90 (Hsp87 and Hsp85) is likely to be modulated by Ca2+/CaM under normal and thermal stress conditions. The likely implications of these findings are discussed.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) 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 (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/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, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/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 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.