Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

Variants in ASPH cause exertional heat illness and are associated with malignant hyperthermia susceptibility.

Exertional heat illness (EHI) and malignant hyperthermia (MH) are life threatening conditions associated with muscle breakdown in the setting of triggering factors including volatile anesthetics, exercise, and high environmental temperature. To identify new genetic variants that predispose to EHI and/or MH, we performed genomic sequencing on a cohort with EHI/MH and/or abnormal caffeine-halothane contracture test. In five individuals, we identified rare, pathogenic heterozygous variants in ASPH, a gene encoding junctin, a regulator of excitation-contraction coupling. We validated the pathogenicity of these variants using orthogonal pre-clinical models, CRISPR-edited C2C12 myotubes and transgenic zebrafish. In total, we demonstrate that ASPH variants represent a new cause of EHI and MH susceptibility.

How mutations in RYR1 that cause malignant hyperthermia increase RYR1 sensitivity to activators.

New electron cryomicroscopy structures of RYR1 show that mutations associated with Malignant Hyperthermia drive conformational changes in the cytoplasmic domains of the closed channel to more closely resemble those of the open channel.

Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression.

Ryanodine receptor 1-related disorders: an historical perspective and proposal for a unified nomenclature.

The RYR1 gene, which encodes the sarcoplasmic reticulum calcium release channel or type 1 ryanodine receptor (RyR1) of skeletal muscle, was sequenced in 1988 and RYR1 variations that impair calcium homeostasis and increase susceptibility to malignant hyperthermia were first identified in 1991. Since then, RYR1-related myopathies (RYR1-RM) have been described as rare, histopathologically and clinically heterogeneous, and slowly progressive neuromuscular disorders. RYR1 variants can lead to dysfunctional RyR1-mediated calcium release, malignant hyperthermia susceptibility, elevated oxidative stress, deleterious post-translational modifications, and decreased RyR1 expression. RYR1-RM-affected individuals can present with delayed motor milestones, contractures, scoliosis, ophthalmoplegia, and respiratory insufficiency.Historically, RYR1-RM-affected individuals were diagnosed based on morphologic features observed in muscle biopsies including central cores, cores and rods, central nuclei, fiber type disproportion, and multi-minicores. However, these histopathologic features are not always specific to RYR1-RM and often change over time. As additional phenotypes were associated with RYR1 variations (including King-Denborough syndrome, exercise-induced rhabdomyolysis, lethal multiple pterygium syndrome, adult-onset distal myopathy, atypical periodic paralysis with or without myalgia, mild calf-predominant myopathy, and dusty core disease) the overlap among diagnostic categories is ever increasing. With the continuing emergence of new clinical subtypes along the RYR1 disease spectrum and reports of adult-onset phenotypes, nuanced nomenclatures have been reported (RYR1- [related, related congenital, congenital] myopathies). In this narrative review, we provide historical highlights of RYR1 research, accounts of the main diagnostic disease subtypes and propose RYR1-related disorders (RYR1-RD) as a unified nomenclature to describe this complex and evolving disease spectrum.

MyoSight-semi-automated image analysis of skeletal muscle cross sections.

BACKGROUND: Manual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses. RESULTS: We describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx). CONCLUSIONS: MyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.

Adaptive thermogenesis enhances the life-threatening response to heat in mice with an Ryr1 mutation.

Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to Ca2+ driven increases in muscle metabolism. Lactate increases brown adipogenesis in both mouse and human brown preadipocytes. This study suggests that simple lifestyle modifications such as avoiding extreme temperatures and maintaining thermoneutrality could decrease the risk of life-threatening responses to heat and exercise in individuals with RYR1 pathogenic variants.

A chemical chaperone improves muscle function in mice with a RyR1 mutation.

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.

Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial ß-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle.

Ca(2+) permeation and/or binding to CaV1.1 fine-tunes skeletal muscle Ca(2+) signaling to sustain muscle function.

BACKGROUND: Ca(2+) influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca(2+) permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed. METHODS: We generated a mouse with a Ca(2+) binding and/or permeation defect in the voltage-dependent Ca(2+) channel, CaV1.1, and used Ca(2+) imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca(2+) imaging techniques to define pathways modulated by Ca(2+) binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles. RESULTS: Using mice with a pore mutation in CaV1.1 required for Ca(2+) binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca(2+) stores during sustained activity. Decreases in these Ca(2+)-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca(2+) binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers. CONCLUSIONS: While not essential for excitation-contraction coupling, Ca(2+) binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 µg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

Mice with RyR1 mutation (Y524S) undergo hypermetabolic response to simvastatin.

BACKGROUND: Statins are widely used drugs for the treatment of hyperlipidemia. Though relatively safe, some individuals taking statins experience rhabdymyolysis, muscle pain, and cramping, a condition termed statin-induced myopathy (SIM). To determine if mutations in the skeletal muscle calcium (Ca2+) release channel, ryanodine receptor type 1 (RyR1), enhance the sensitivity to SIM we tested the effects of simvastatin, the statin that produces the highest incidence of SIM in humans, in mice with a mutation (Y524S, 'YS') in RyR1. This mutation is associated with malignant hyperthermia in humans. Exposure of mice with the YS mutation to mild elevations in environmental temperature produces a life-threatening hypermetabolic response (HMR) that is characterized by increased oxygen consumption (VO2), sustained muscle contractures, rhabdymyolysis, and elevated core body temperature. METHODS: We assessed the ability of simvastatin to induce a hypermetabolic response in the YS mice using indirect calorimetry and to alter Ca2+ release via RyR1 in isolated flexor digitorum brevis (FDB) fibers from WT and YS mice using fluorescent Ca2+ indicators. We also tested the ability of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) to protect against the simvastatin effects. RESULTS: An acute dose of simvastatin triggers a hypermetabolic response in YS mice. In isolated YS muscle fibers, simvastatin triggers an increase in cytosolic Ca2+ levels by increasing Ca2+ leak from the sarcoplasmic reticulum (SR). With higher simvastatin doses, a similar cytosolic Ca2+ increase occurs in wild type (WT) muscle fibers. Pre-treatment of YS and WT mice with AICAR prevents the response to simvastatin. CONCLUSIONS: A mutation in RyR1 associated with malignant hyperthermia increases susceptibility to an adverse response to simvastatin due to enhanced Ca2+ release from the sarcoplasmic reticulum, suggesting that RyR1 mutations may underlie enhanced susceptibility to statin-induced myopathies. Our data suggest that AICAR may be useful for treating statin myopathies.

Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia.

Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell 'cores'. Imaging cytosolic and intra-SR [Ca2+] in muscle cells of heterozygous YS mice we determined Ca2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca2+] gradient) and SR Ca2+ buffering power (B). In YS cells resting [Ca2+]SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca2+]SR(t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca2+-dependent inactivation (CDI). The [Ca2+] transients showed abnormal 'breaks', decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibres with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca2+]SR and alters SR Ca2+ buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca2+ control that defines a fulminant MH event.

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation.

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca(2+) leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca(2+)-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca(2+) concentrations. If unchecked, the temperature-driven increases in resting Ca(2+) concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.

Ryanodine receptors: structure, expression, molecular details, and function in calcium release.

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (> 2MDa) and exist as three mammalian isoforms (RyR 1-3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.

Effect of prior exercise on thermal sensitivity of malignant hyperthermia-susceptible muscle.

Malignant hyperthermia (MH) episodes may occur upon exposure to halogenated anesthetics, during resistance and endurance exercise, and in response to thermal stress. The purpose of this study was to investigate the effects of prior eccentric and concentric (i.e., wheel running) exercise on the thermal sensitivity of isolated MH-susceptible mouse muscle (RyR1(Y522S/wt)). Eccentric, but not concentric, exercise attenuated the thermal sensitivity of MH-susceptible muscle.

Role of essential oils in control of Rhizoctonia damping-off in tomato with bioactive Monarda herbage.

Plants in the genus Monarda produce complex essential oils that contain antifungal compounds. The objectives of this research were to identify selections of monarda that reduce Rhizoctonia damping-off of tomato, and to determine relationships between essential oil composition of 13 monarda herbages (dried and ground leaves) and disease suppression. Herbages were grouped into five chemotypes, based on essential oil composition and effective concentrations for reducing growth by 50% for Rhizoctonia solani. Replicated and repeated disease control assays were conducted with monarda herbages in greenhouse medium, with or without Rhizoctonia. Percent survival, seedling height, and stem diameter were evaluated at 8 weeks. Survival, seedling height, and stem diameter in herbage-only treatments were not different from the control (no-herbage, no-pathogen) for most herbage treatments. In the pathogen control (no-herbage + Rhizoctonia), seedling survival was 10% that of the control. In pathogen-infested media, seedling survival ranged from 65 to 80% for treatments with thymol chemotypes and 55 to 65% for carvacrol chemotypes. Effective control of Rhizoctonia damping-off was correlated with phenolic monoterpenes; herbages classified as carvacrol chemotypes effectively protected tomato seedlings from Rhizoctonia damping-off disease without phytotoxicity. This study provides evidence that monarda herbages have potential as growing media amendments for control of Rhizoctonia damping-off disease.

Characterization and temporal development of cores in a mouse model of malignant hyperthermia.

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

Determinants in CaV1 channels that regulate the Ca2+ sensitivity of bound calmodulin.

Calmodulin binds to IQ motifs in the alpha(1) subunit of Ca(V)1.1 and Ca(V)1.2, but the affinities of calmodulin for the motif and for Ca(2+) are higher when bound to Ca(V)1.2 IQ. The Ca(V)1.1 IQ and Ca(V)1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca(V)1.1 IQ and compared it with that of calmodulin bound to Ca(V)1.2 IQ. Four methionines in Ca(2+)-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca(V)1.1 and Tyr-1675 of Ca(V)1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca(V)1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca(V)1.2 contribute significantly to the difference in the Ca(2+) affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca(2+)-bound and one lobe Ca(2+)-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca(V)1.2.

Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+* calmodulins.

Voltage-dependent calcium channels (Ca(V)) open in response to changes in membrane potential, but their activity is modulated by Ca(2+) binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca(V)1.2 alpha(1) subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca(2+).CaM in 2 distinct binding modes. The structure of the Ca(V)1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca(2+).CaMs interacting with the pre-IQ regions and a canonical Ca(V)1-IQ-Ca(2+).CaM complex. Native Ca(V)1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca(2+)-dependent inactivation of heterologously expressed Ca(V)1.2 channels.