Inactivation Of Calcium Channels Research Articles

Local Control Model of a Human Ventricular Myocyte: An Exploration of Frequency-Dependent Changes and Calcium Sparks.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted "nonconducting" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca2+-sensingprotein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca2+-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Cav1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

Novel CACNA1C R511Q mutation, located in domain Ⅰ-Ⅱ linker, causes non-syndromic type-8 long QT syndrome.

BackgroundGain-of-function mutations in CACNA1C encoding Cav1.2 cause syndromic or non-syndromic type-8 long QT syndrome (LQTS) (sLQT8 or nsLQT8). The cytoplasmic domain (D)Ⅰ-Ⅱ linker in Cav1.2 plays a pivotal role in calcium channel inactivation, and mutations in this site have been associated with sLQT8 (such as Timothy syndrome) but not nsLQT8.ObjectiveSince we identified a novel CACNA1C mutation, located in the DⅠ-Ⅱ linker, associated with nsLQTS, we sought to reveal its biophysical defects.MethodsTarget panel sequencing was employed in 24 genotype-negative nsLQTS probands (after Sanger sequencing) and three family members. Wild-type (WT) or R511Q Cav1.2 was transiently expressed in tsA201 cells, then whole-cell Ca2+ or Ba2+ currents (ICa or IBa) were recorded using whole-cell patch-clamp techniques.ResultsWe identified two CACNA1C mutations, a previously reported R858H mutation and a novel R511Q mutation located in the DⅠ-Ⅱ linker. Four members of one nsLQTS family harbored the CACNA1C R511Q mutation. The current density and steady-state activation were comparable to those of WT-ICa. However, persistent currents in R511Q-ICa were significantly larger than those of WT-ICa (WT at +20 mV: 3.3±0.3%, R511Q: 10.8±0.8%, P<0.01). The steady-state inactivation of R511Q-ICa was weak in comparison to that of WT-ICa at higher prepulse potentials, resulting in increased window currents in R511Q-ICa. Slow component of inactivation of R511Q-ICa was significantly delayed compared to that of WT-ICa (WT-tau at +20 mV: 81.3±3.3 ms, R511Q-tau: 125.1±5.0 ms, P<0.01). Inactivation of R511Q-IBa was still slower than that of WT-IBa, indicating that voltage-dependent inactivation (VDI) of R511Q-ICa was predominantly delayed.ConclusionsDelayed VDI, increased persistent currents, and increased window currents of R511Q-ICa cause nsLQT8. Our data provide novel insights into the structure-function relationships of Cav1.2 and the pathophysiological roles of the DⅠ-Ⅱ linker in phenotypic manifestations.

STAC proteins inhibit calcium and voltage dependent inactivation of L-type calcium channels

Upon depolarization, voltage-gated calcium channels (VGCC) open but then undergo inactivation. Two processes govern VGCC inactivation: calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI). Recently, the members of the STAC family of adaptor proteins have been shown to inhibit CDI of L-type calcium channels, by binding to the C-terminal domain. Here, we demonstrate that STAC proteins also inhibit the VDI of CaV1.2 and CaV1.3. Remarkably, using a STAC mutant, we could demonstrate that the inhibition of VDI relies on the same interaction, between STAC and the calcium channel, as CDI.

De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca2+ regulation

Sudden unexplained death in childhood (SUDC) is an understudied problem. Whole-exome sequence data from 124 "trios" (decedent child, living parents) was used to test for excessive de novo mutations (DNMs) in genes involved in cardiac arrhythmias, epilepsy, and other disorders. Among decedents, nonsynonymous DNMs were enriched in genes associated with cardiac and seizure disorders relative to controls (odds ratio = 9.76, P = 2.15 × 10-4). We also found evidence for overtransmission of loss-of-function (LoF) or previously reported pathogenic variants in these same genes from heterozygous carrier parents (11 of 14 transmitted, P = 0.03). We identified a total of 11 SUDC proband genotypes (7 de novo, 1 transmitted parental mosaic, 2 transmitted parental heterozygous, and 1 compound heterozygous) as pathogenic and likely contributory to death, a genetic finding in 8.9% of our cohort. Two genes had recurrent missense DNMs, RYR2 and CACNA1C Both RYR2 mutations are pathogenic (P = 1.7 × 10-7) and were previously studied in mouse models. Both CACNA1C mutations lie within a 104-nt exon (P = 1.0 × 10-7) and result in slowed L-type calcium channel inactivation and lower current density. In total, six pathogenic DNMs can alter calcium-related regulation of cardiomyocyte and neuronal excitability at a submembrane junction, suggesting a pathway conferring susceptibility to sudden death. There was a trend for excess LoF mutations in LoF intolerant genes, where ≥1 nonhealthy sample in denovo-db has a similar variant (odds ratio = 6.73, P = 0.02); additional uncharacterized genetic causes of sudden death in children might be discovered with larger cohorts.

Robust switches in thalamic network activity require a timescale separation between sodium and T-type calcium channel activations.

Switches in brain states, synaptic plasticity and neuromodulation are fundamental processes in our brain that take place concomitantly across several spatial and timescales. All these processes target neuron intrinsic properties and connectivity to achieve specific physiological goals, raising the question of how they can operate without interfering with each other. Here, we highlight the central importance of a timescale separation in the activation of sodium and T-type calcium channels to sustain robust switches in brain states in thalamic neurons that are compatible with synaptic plasticity and neuromodulation. We quantify the role of this timescale separation by comparing the robustness of rhythms of six published conductance-based models at the cellular, circuit and network levels. We show that robust rhythm generation requires a T-type calcium channel activation whose kinetics are situated between sodium channel activation and T-type calcium channel inactivation in all models despite their quantitative differences.

Aborted Sudden Death Due to Severe Ventricular Arrhythmia in Timothy Syndrome

Abstract Timothy Syndrome is a rare autosomal dominant multisystem genetic condition. The CACNA1C gene, codifier of the CaV1.2 calcium channel, is affected, resulting in the loss of voltage-dependent calcium channel inactivation. Relevant clinical characteristics: (1) corrected QT interval greater than 480ms; (2) syndactyly. Death often occurs during childhood, and results from ventricular tachyarrhythmias. This study presents the case of a female newborn who suffered a cardiorespiratory arrest, secondary to ventricular arrhythmia. A prolonged QT interval, combined with 2:1 AV block, [...]

Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

Capsaicin Causes Vasorelaxation of Rat Aorta through Blocking of L-type Ca2+ Channels and Activation of CB1 Receptors.

The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin.

The effect of MD1 on potassium and L-type calcium current of cardiomyocytes from high-fat diet mice

ABSTRACT Myeloid differentiation protein 1 (MD1) is exerted an anti-arrhythmic effect in obese mice. Therefore, we sought to clarify whether MD1 can alter the electrophysiological remodeling of cardiac myocytes from obese mice by regulating voltage-gated potassium current and calcium current. MD1 knock-out (KO) and wild type (WT) mice were given a high-fat diet (HFD) for 20 weeks, starting at the age of 6 weeks. The potential electrophysiological mechanisms were estimated by whole-cell patch-clamp and molecular analysis. After 20-week HFD feeding, action potential duration (APD) from left ventricular myocytes of MD1-KO mice revealed APD20, APD50, and APD90 were profoundly enlarged. Furthermore, HFD mice showed a decrease in the fast transient outward potassium currents (Ito,f), slowly inactivating potassium current (IK, slow), and inward rectifier potassium current (IK1). Besides, HFD-fed mice showed that the current density of ICaL was significantly lower, and the haft inactivation voltage was markedly shifted right. These HFD induced above adverse effects were further exacerbated in KO mice. The mRNA expression of potassium ion channels (Kv4.2, Kv4.3, Kv2.1, Kv1.5, and Kir2.1) and calcium ion channel (Cav1.2) was markedly decreased in MD1-KO HFD-fed mice. MD1 deletion led to down-regulated potassium currents and slowed inactivation of L-type calcium channel in an obese mice model.

Влияние соединения ФС-ЛХТ-317 на активность NMDA-рецепторов в смешанной нейроглиальной культуре гиппокампа крысы

В опытах на нейроглиальной культуре клеток гиппокампа крыс Spraque Dawley методом флуоресцентной визуализации оценили влияние соединения ФС-ЛХТ-317 (магния бис-ацетаминоэтансульфоната), обладающего церебропротекторным действием, на естественную инактивацию кальциевых каналов NMDA-рецепторов. Установлено, что это вещество в течение 0,2 – 0,4 с после аппликации оказывает ингибиторный эффект, направленный на подавление активности NMDA-рецепторов. Данный эффект формируется в диапазоне концентраций от 1 до 10 мМ и является обратимым.

Effects of exendin-4 on colonic motility in rats and its underlying mechanism.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists modulate gastrointestinal motility; however, the effects of GLP-1R agonists on colonic motility are still controversial, and the molecular mechanism is unclear. Exendin-4 shares 53% homology with GLP-1 and is a full agonist of GLP-1R. In this study, our aims were to explore the role and mechanism of exendin-4 in isolated rat colonic tissues and cells. An organ bath system was used to examine the spontaneous contractions of smooth muscle strips. The whole-cell patch-clamp technique was used to investigate the currents of L-type voltage-dependent calcium channels and large conductance Ca2+ -activated K+ (BKCa ) channels in smooth muscle cells. Exendin-4 decreased both the amplitude and frequency of spontaneous contractions of smooth muscle strips in a concentration-dependent manner. The inhibitory effect was completely blocked by exendin-4(9-39), a GLP-1R antagonist. Moreover, this effect was partially abolished by tetrodotoxin (TTX), a blocker of neuronal voltage-dependent Na+ channels, Nω-Nitro-l-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor, apamin, an inhibitor of small-conductance Ca2+ -activated K+ (SK) channels. Whole-cell patch-clamp recordings revealed that exendin-4 inhibited the peak current of L-type calcium channels in colonic smooth muscle cells, but did not change the shape of the current-voltage (I-V) curves. The steady-state activation and steady-state inactivation of L-type calcium channels were not affected. Likewise, BKCa currents were significantly inhibited by exendin-4. Exendin-4 indirectly inhibits colonic muscle activity via a nitrergic and a purinergic neural pathway through NO and ATP release and inhibits L-type voltage-dependent calcium channels and BKCa channels in smooth muscle cells.

Treatment of calmodulinopathy with verapamil

Pathological variants in genes encoding calmodulin are associated with severe clinical presentations, including recurrent ventricular fibrillation and sudden death. Beta-receptor antagonists (beta-blockers) and sodium-channel antagonists have been reported as pharmacotherapies...

Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation

Inactivation of L-type calcium channel (Cav1.2) is an important determinant of the length of the cardiac action potential. Here, we report a key role of the voltage-sensing segment IS4 in Cav1.2 inactivation. Neutralization of IS4 charges gradually shifted the steady-state inactivation curve on the voltages axis from 5.1 ± 3.7 mV in single point mutant IS4(K1Q) to −26.7 ± 1.3 mV in quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) compared to wild-type (WT) and accelerated inactivation. The slope factor of the Boltzmann curve of inactivation was decreased from 17.4 ± 3.5 mV (IS4(K1Q)) to 6.2 ± 0.7 mV (IS4(K1Q/R2Q/R3Q/R4Q)). Neutralizations of single or multiple charges in IIS4 and IIIS4 did not significantly affect the time course of inactivation. Neutralization of individual IVS4 charges shifted the inactivation curve between 17.4 ± 1.7 mV (IVS4(R2Q)) and −4.6 ± 1.4 mV (IVS4(R4Q)) on the voltage axis and affected the slope of the inactivation curves (IVS4(R2Q): 10.2 ± 1.2 mV, IVS4(R4Q): 9.7 ± 0.7 mV and IVS4(K5Q): 8.1 ± 0.7 mV vs WT: 14.1 ± 0.8 mV). IS4(K1Q) attenuated while IS4(K1Q/R2Q/R3Q) and IS4(K1Q/R2Q/R4Q/R3Q) enhanced the development of inactivation. Shifts in the voltage dependence of inactivation curves induced by IS4 neutralizations significantly correlated with shifts of the voltage dependence of channel activation (r = 0.95, p < 0.01) indicating that IS4 movement is not only rate limiting for activation but also initiates inactivation. The paradoxical decrease of the slope factor of the steady-state inactivation and acceleration of inactivation kinetics upon charge neutralization in segment IS4 may reflect the loss of stabilizing interactions of arginines and lysine with surrounding residues.

The role of L-type calcium channels in mouse oocyte maturation, activation and early embryonic development

Calcium ion fluctuation is closely related to the transformation of cell cycle. However, little is known about the function of L-type calcium channel in mammalian oocyte and embryo development. We thus studied the roles of L-type calcium channel in mouse oocyte meiotic maturation, parthenogenetic activation and early embryonic development. We used the antagonist Amlodipine to block L-type calcium channel. Oocytes or zygotes were cultured to different time points with 0 μM, 10 μM, 30 μM and 50 μM Amlodipine. Then we checked the rate of first polar body extrusion, spindle formation, asymmetric division parthenogenetic activation and early embryo cleavage. The results showed that Amlodipine treatment did not affect germinal vesicle breakdown, but caused disruption of cytoskeleton organization, symmetric division, formation of mature oocytes with a large polar body, or reduced the first polar body extrusion, depending on its concentrations. Amlodipine treatment also resulted in decreased parthenogenetic activation and arrested early embryonic development. Overall, these data suggest that proper function of L-type calcium channel is critical for oocyte maturation, activation, and early embryonic development.

Auditory processing enhancements in the TS2-neo mouse model of Timothy Syndrome, a rare genetic disorder associated with autism spectrum disorders.

Timothy syndrome (TS) is a rare genetic disorder caused by a single de novo missense mutation to the 8A exon of CACNA1C gene, which codes for the voltage-gated L-type Ca2+ channel (Cav1.2). TS is strongly associated with cardiac arrthytmias, autism spectrum disorders (ASDs), and neurological dysfunction such as language impairments, seizures, and intellectual disability. A genetically engineered knock-in mouse with a heterogeneous TS2 (G406R) mutation in the L-type calcium channel containing a neomycin resistance cassette was developed to study ASD-like behaviors. This mouse model (TS2-neo) provides us with a platform to investigate the role of calcium channel inactivation and calcium signaling related to brain development and ASD. The purpose of the current study was to behaviorally characterize TS2-neo mice by assessing their performance on a wide variety of behavioral paradigms, including replication of findings that support the TS2-neo as a valid platform for studying ASD-like behavior. In addition to examining core social and repetitive anomalies, we sought to focus on basic perceptual processing in the auditory domain. Results indicate that the loss of Cav1.2 inactivation in this mouse model results in deviant social and repetitive behaviors as well as poor sensorimotor learning. Additionally, TS2-neo mice display superior performance on both an embedded tone and silent gap discrimination task for short-duration acoustic stimuli. These findings parallel the low-level auditory enhancements observed within the ASD clinical population. Additionally, structural anomalies were seen for mutant mice in some white matter tracts and in the medial geniculate nucleus (MGN). Co-occurrence of these findings suggests that aberrant MGN morphology may be related to enhanced auditory processing phenotype as seen in ASD.

Structural Characterization of Calmodulin Disease Mutations

Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in the propagation of intracellular calcium signals and the regulation of events ranging from muscle contraction to cell excitability. The human genome contains three CaM genes (CALM 1-3) which encode for protein with identical primary sequences. Despite the redundancy of CaM, single missense mutations in even one of the six alleles are associated with disease phenotypes such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and early-onset severe long QT syndrome (esLQT). CPVT can lead to stress- and exercise-induced arrhythmias and sudden cardiac death; esLQT is characterized by a prolonged QT interval which can also result in ventricular fibrillation. Despite the devastating genetic disorders associated with CaM mutations, the molecular mechanisms by which these mutations manifest into dominant disease phenotypes have yet to be elucidated. Here, we present the crystal structures of several CaM disease mutants, one of which represents a novel CaM conformation not previously characterized. Significant structural changes are observed in both EF-hands III and IV of the C-lobe. In particular, the mutation disrupts the calcium coordination network in EF-hand III and results in abolished calcium binding, leading to CaM that resembles an intermediate between the Ca2+-CaM and apo-CaM states. In contrast, structures of other disease mutants revealed CaM conformations that closely resemble the wild type structure, with little positional shift in the EF-hand helices of either the N- or C-lobe. These structures can help explain the diverse effects of CaM mutations and the associated disease mechanisms, especially as CaM mutations have differential effects on the function of ryanodine receptor 2 and calcium-dependent inactivation of L-type calcium channels.