Slow Activation Kinetics Research Articles

Lipophilic compounds restore function to neurodevelopmental-associated KCNQ3 mutations

A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss– and gain–of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel’s pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

Differential pathogenetic mechanisms of mutations in helix 2 and helix 6 of rhodopsin

Variants in rhodopsin (RHO) have been linked to autosomal dominant congenital stationary night blindness (adCSNB), which affects the ability to see in dim light, and the pathogenetic mechanism is still not well understood. In this study we report two novel RHO variants found in adCSNB families, p.W265R and p.A269V, that map in the sixth transmembrane domain of RHO protein. We applied in silico molecular simulation and in vitro biochemical and molecular studies to characterize the two new variants and compare the molecular determinants to two previously characterized adCSNB variants, p.G90D and p.T94I, that map in the second transmembrane domain of the RHO protein. We demonstrate that W265R and A269V cause constitutive activation of RHO with light-independent G protein coupling and impaired binding to arrestin. Differently, G90D and T94I are characterized by slow kinetics of RHO activation and deactivation. This study provides new evidence on the differential contribution of transmembrane α-helixes two and six to the interaction with intracellular transducers of RHO and mutations in these helixes result in a similar phenotype in patients but with distinct molecular effects.

Novel insights into the modulation of the voltage-gated potassium channel KV1.3 activation gating by membrane ceramides

Membrane lipids extensively modulate the activation gating of voltage-gated potassium channels (KV), however, much less is known about the mechanisms of ceramide and glucosylceramide actions including which structural element is the main intramolecular target and whether there is any contribution of indirect, membrane biophysics-related mechanisms to their actions. We used two-electrode voltage-clamp fluorometry capable of recording currents and fluorescence signals to simultaneously monitor movements of the pore domain (PD) and the voltage sensor domain (VSD) of the KV1.3 ion channel after attaching an MTS-TAMRA fluorophore to a cysteine introduced into the extracellular S3-S4 loop of the VSD. We observed rightward shifts in the conductance-voltage (G-V) relationship, slower current activation kinetics, and reduced current amplitudes in response to loading the membrane with C16-ceramide (Cer) or C16-glucosylceramide (GlcCer). When analyzing VSD movements, only Cer induced a rightward shift in the fluorescence signal-voltage (F-V) relationship and slowed fluorescence activation kinetics, whereas GlcCer exerted no such effects. These results point at a distinctive mechanism of action with Cer primarily targeting the VSD, while GlcCer only the PD of KV1.3. Using environment-sensitive probes and fluorescence-based approaches, we show that Cer and GlcCer similarly increase molecular order in the inner, hydrophobic regions of bilayers, however, Cer induces a robust molecular reorganization at the membrane-water interface. We propose that this unique ordering effect in the outermost membrane layer in which the main VSD rearrangement involving an outward sliding of the top of S4 occurs can explain the VSD targeting mechanism of Cer, which is unavailable for GlcCer.

Abstract PO3-23-10: Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations

Abstract CDK4/6 inhibitors (CDK4/6i) have significantly improved the prognosis of patients with metastatic hormone receptor-positive breast cancer. However, the development of drug resistance leads to the discontinuation of CDK4/6i treatment. The potential benefits of maintaining CDK4/6i treatment in the context of resistance and their underlying molecular mechanisms remain elusive. Here we show that despite tumor progression, continued treatment with CDK4/6i notably extends the G1 phase in drug-resistant cells, thereby slowing their growth. Our single-cell data reveal that the duration of the G1 phase in these resistant cells is approximately tripled under continuous CDK4/6i treatment compared to conditions with CDK4/6i withdrawal, while the durations of the S and G2 phases remain comparable. The maintenance of CDK4/6i induces an alternative inactivation of the retinoblastoma (Rb) protein in drug-resistant cells through its post-translational degradation. However, this alternative form of Rb inactivation is ineffective and thus induces slow activation kinetics of E2F transcription factors, significantly delaying G1 progression. The beneficial effect of maintaining CDK4/6i treatment was further validated in breast cancer xenograft models. Furthermore, our findings suggest that given the spectrum of various CDK4/6i, switching between different CDK4/6i types after the emergence of drug resistance may further delay tumor progression. This study provides new mechanistic insights into the maintenance of CDK4/6i treatment despite the development of drug resistance. Citation Format: Jessica Armand, Sungsoo Kim, Hee Won Yang. Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-10.

De novo variants in KCNA3 cause developmental and epileptic encephalopathy.

Variants in several potassium channel genes, including KCNA1 and KCNA2, cause Developmental and Epileptic Encephalopathies (DEEs). We investigated whether variants in KCNA3, another mammalian homologue of the Drosophila shaker family and encoding for Kv1.3 subunits, can cause DEE. Genetic analysis of study individuals was performed by routine exome or genome sequencing, usually of parent-offspring trios. Phenotyping was performed via a standard clinical questionnaire. Currents from wild-type and/or mutant Kv1.3 subunits were investigated by whole-cell patch-clamp upon their heterologous expression. Fourteen individuals, each carrying a de novo heterozygous missense variant in KCNA3, were identified. Most (12/14; 86%) had DEE with marked speech delay with or without motor delay, intellectual disability, epilepsy, and autism spectrum disorder. Functional analysis of Kv1.3 channels carrying each variant revealed heterogeneous functional changes, ranging from "pure" loss-of-function (LoF) effects due to faster inactivation kinetics, depolarized voltage-dependence of activation, slower activation kinetics, increased current inactivation, reduced or absent currents with or without dominant-negative effects, to "mixed" loss- and gain-of-function (GoF) effects. Compared to controls, Kv1.3 currents in lymphoblasts from 1 of the proband displayed functional changes similar to those observed upon heterologous expression of channels carrying the same variant. The antidepressant drug fluoxetine inhibited with similar potency the currents from wild-type and 1 of the Kv1.3 GoF variant. We describe a novel association of de novo missense variants in KCNA3 with a human DEE, and provide evidence that fluoxetine might represent a potential targeted treatment for individuals carrying variants with significant GoF effects. ANN NEUROL 2024;95:365-376.

Bacterial sodium channels as gene therapy for cardiac arrhythmia: slow (activation and inactivation kinetics) and steady wins the race.

Bacterial sodium channels as gene therapy for cardiac arrhythmia: slow (activation and inactivation kinetics) and steady wins the race.

High-Throughput Screening of Nitrogen Reduction Reaction on Single Atom@1T'-MoS2

Electrochemical synthesis of ammonia through nitrogen reduction reaction (NRR) is cost-effective and energy-effective compared to the conventional Haber-Bosch process. However, the slow activation kinetics of N2 hindered the development of active and selective catalysts. In this work, we showed that transition metal (TM) single atoms anchored on monolayer molybdenum disulfide (MoS2) with 1T phase could promote its phase transition to 1T' due to broken surface symmetry induced by TM atoms. By combining the high-throughput screening with DFT calculations in transition metals, two TM elements (V, Ti) anchored on 1T'-MoS2 were proved to be promising candidates in N2 fixation. The V and Ti single-atom catalyst exhibits low Gibbs free energy barrier, and Ti single-atom catalyst shows the highest selectivity towards NH3. Moreover, the stability of single-atom TM@1T'-MoS2 was measured with the ab initio molecular dynamics (AIMD) simulations. The density of states (DOS) and surface charge analysis were conducted to reveal the origin of improved activities. Our results pave the way to design more innovative single-atom catalysts for high-performance electrocatalytic NRR.

Metal–Organic Frameworks Encaged Ru Single Atoms for Rapid Acetylene Harvest and Activation in Hydrochlorination

Ruthenium (Ru)-based catalysts have been candidates in hydrochlorination for vinyl chloride monomer (VCM) production, yet they are limited by efficient acetylene (C2H2) utilization. The strong adsorption performance of HCl can deactivate Ru active sites which resulted in weak C2H2 adsorption and slow activation kinetics. Herein, we designed a channel that employed metal-organic framework (MOF)-encaged Ru single atoms to achieve rapid adsorption and activation of C2H2. Low-Ru (∼0.5 wt %) single-atom catalysts (named Ru-NC@MIL) were assembled by hydrogen-bonding nanotraps (the H-C≡C-Hδ+···Oδ- interactions between C2H2 and carboxylate groups/furan rings). Results confirmed that C2H2 could easily enter the encapsulation channels in an optimal mode perpendicular to the channel with a potential energy of 42.3 kJ/mol. The harvested C2H2 molecules can be quickly passed to Ru-N4 active sites for activation by stretching the length of carbon-carbon triple bonds (C≡C) to 1.212 Å. Such a strategy guaranteed >99% C2H2 conversion efficiency and >99% VCM selectivity. Moreover, a stable long-term (>150 h) catalysis with high efficiency (∼0.85 kgvcm/h/kgcat.) and a low deactivation constant (0.001 h-1) was also achieved. This work provides an innovative strategy for precise C2H2 adsorption and activation and guidance for designing multi-functional Ru-based catalysts.

Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.

Derivatives of 2-aminobenzimidazole potentiate ASIC open state with slow kinetics of activation and desensitization.

The pharmacology of acid-sensitive ion channels (ASICs) is diverse, but potent and selective modulators, for instance for ASIC2a, are still lacking. In the present work we studied the effect of five 2-aminobenzimidazole derivatives on native ASICs in rat brain neurons and recombinant receptors expressed in CHO cells using the whole-cell patch clamp method. 2-aminobenzimidazole selectively potentiated ASIC3. Compound Ru-1355 strongly enhanced responses of ASIC2a and caused moderate potentiation of native ASICs and heteromeric ASIC1a/ASIC2a. The most active compound, Ru-1199, caused the strongest potentiation of ASIC2a, but also potentiated native ASICs, ASIC1a and ASIC3. The potentiating effects depended on the pH and was most pronounced with intermediate acidifications. In the presence of high concentrations of Ru-1355 and Ru-1199, the ASIC2a responses were biphasic, the initial transient currents were followed by slow component. These slow additional currents were weakly sensitive to the acid-sensitive ion channels pore blocker diminazene. We also found that sustained currents mediated by ASIC2a and ASIC3 are less sensitive to diminazene than the peak currents. Different sensitivities of peak and sustained components to the pore-blocking drug suggest that they are mediated by different open states. We propose that the main mechanism of action of 2-aminobenzimidazole derivatives is potentiation of the open state with slow kinetics of activation and desensitization.

STAC3 determines the slow activation kinetics of CaV 1.1 currents and inhibits its voltage-dependent inactivation.

The skeletal muscle CaV 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium-dependent inactivation (CDI) of L-typecalcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV 1.3 currents. When coexpressed in CaV 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with CaV 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports CaV 1.1 functional expression and EC coupling, although at a reduced extent compared to wild-type STAC3. Importantly, STAC3-ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV 1.1 currents, suggesting that STAC3 determines the slow CaV 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3-ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage-dependent inactivation (VDI) of CaV 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.

Complex effects on CaV2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder

P/Q-type Ca2+ currents mediated by CaV2.1 channels are essential for active neurotransmitter release at neuromuscular junctions and many central synapses. Mutations in CACNA1A, the gene encoding the principal CaV2.1 α1A subunit, cause a broad spectrum of neurological disorders. Typically, gain-of-function (GOF) mutations are associated with migraine and epilepsy while loss-of-function (LOF) mutations are causative for episodic and congenital ataxias. However, a cluster of severe CaV2.1 channelopathies have overlapping presentations which suggests that channel dysfunction in these disorders cannot always be defined bimodally as GOF or LOF. In particular, the R1667P mutation causes focal seizures, generalized hypotonia, dysarthria, congenital ataxia and, in one case, cerebral edema leading ultimately to death. Here, we demonstrate that the R1667P mutation causes both channel GOF (hyperpolarizing voltage-dependence of activation, slowed deactivation) and LOF (slowed activation kinetics) when expressed heterologously in tsA-201 cells. We also observed a substantial reduction in Ca2+ current density in this heterologous system. These changes in channel gating and availability/expression manifested in diminished Ca2+ flux during action potential-like stimuli. However, the integrated Ca2+ fluxes were no different when normalized to tail current amplitude measured upon repolarization from the reversal potential. In summary, our findings indicate a complex functional effect of R1667P and support the idea that pathological missense mutations in CaV2.1 may not represent exclusively GOF or LOF.

A small and highly sensitive red/far-red optogenetic switch for applications in mammals.

Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR-nuclease-deactivated Cas9 (CRISPR-dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Multifunctional Pt(iv) prodrug candidates featuring the carboplatin core and deferoxamine.

The synergistic combination of the anticancer drug carboplatin and the iron chelator deferoxamine (DFO) served as a foundation for the development of novel multifunctional prodrugs. Hence, five platinum(iv) complexes, featuring the equatorial coordination sphere of carboplatin, and one or two DFO units incorporated at axial positions, were synthesized and characterized using ESI-HRMS, multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy and elemental analysis. Analytical studies demonstrated that the chelating properties of the DFO moiety were not compromised after coupling to the platinum(iv) core. The cytotoxic activity of the compounds was evaluated in monolayer (2D) and spheroid (3D) cancer cell models, derived from ovarian teratocarcinoma (CH1/PA-1), colon carcinoma (SW480) and non-small cell lung cancer (A549). The platinum(iv)-DFO prodrugs demonstrated moderate in vitro cytotoxicity (a consequence of their slow activation kinetics) but with less pronounced differences between intrinsically chemoresistant and chemosensitive cell lines as well as between 2D and 3D models than the clinically used platinum(ii) drug carboplatin.

Domain zipping and unzipping modulates TRPM4's properties in human cardiac conduction disease.

The transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.

Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP2K

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11‐Pbs2‐Hog1 MAPK cascade and the Ssk2/Ssk22‐Pbs2‐Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr‐518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser‐514 and Thr‐518 under optimal osmostress conditions. Mono‐phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2‐Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone‐to‐Hog1 crosstalk in the absence of osmostress. We also report that the rapid‐and‐transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

Domain Zipping and Unzipping Modulates TRPM4's Properties in Human Cardiac Conduction Disease

The transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.

Which CaV1.1 Voltage Sensor(s) Activate RYR1?

The skeletal muscle L-type Ca­V channel is composed by the pore-forming α1s (CaV1.1) in association with α2δ-1, β1a and γ1 subunits in 1:1:1:1 stoichiometry. This large protein complex makes physical contact with Ryanodine (RyR1) channel located in the sarcoplasmic reticulum (SR), conferring voltage-dependence to the Ca2+ release channel, thus coupling electrical excitation with contraction. CaV1.1 channels possess four homologous but non-identical voltage sensing domains (VSDs), which undergo voltage-dependent rearrangements: we propose that one VSD is a stronger candidate than all others to be the sensor responsible for RyR1 voltage-dependent activation. We reconstituted human CaV1.1 complexes (α1s, α2δ-1, β1a, γ1 and stac3) in Xenopus oocytes. We used the voltage clamp fluorometry technique to simultaneously record Ca2+ currents (2mM external Ca2+) and the structural rearrangements of the four individual VSDs. We found that the voltage-dependent activation curves of the four VSDs are spread from −150 to +80mV, all preceding pore opening, with VSD-I and VSD-IV operating at more hyperpolarized potentials (VSD-I Vhalf=-35.6±2.2mV, z=1.0±0.01e0, VSD-IV Vhalf=-64.9±2.7mV, z=0.74±0.02e0) than VSD-II and VSD-III (VSD-II Vhalf=7.2±2.8mV, z=1.6±0.1e0, VSD-III Vhalf=-23.9±3mV, z=1.6±0.04e0). Each VSD exhibited distinct time-dependent properties, with VSD-I and VSD-II displaying slow activation kinetics (VSD-I τ=39.8±2ms, VSD-II τ=132±16ms, at 20mV), while VSD-III and VSD-IV activated rapidly (VSD-III τ=1.8±0.2ms, VSD-IV τ=2±1ms, at 20mV). The operation of the four voltage sensors was then investigated under a physiologically-relevant action potential (AP) clamp (skeletal muscle AP). Within the short AP clamp (∼5ms), no clear structural rearrangements of the slowest sensors (VSD-I and VSD-II) could be observed, while VSD-III and VSD-IV were readily activated. Based on i) kinetics, ii) voltage-dependence, iii) proximity with intracellular II-III loop (known to be a critical element in EC coupling), VSD-III is a strong candidate as sensor conferring voltage-dependent properties to RyR1 and the SR Ca2+ release.

Structural Determinants of Voltage-Gated Calcium Channel Gating Properties

Voltage-gated calcium channels (CaV) control most activity-dependent functions of excitable cells, but the molecular mechanism how they respond to membrane depolarization with specific gating properties remains unknown. CaV channels consist of four homologous but non-identical repeats (I, II, III, IV), each containing a separate voltage-sensing domain (VSD) arranged around the common channel pore. Within each VSD the positive gating charges (K0, R1-R4) in the transmembrane helix S4 sequentially interact with negative counter-charges in helices S2 and S3 to support the movement of the gating charges across the electrical field of the membrane and thus to activate or deactivate the channel. We combined MD simulation and Markov-state models with mutagenesis and electrophysiological analysis to study the structure, free energy levels, and transition times of the activated and resting states of two distinct voltage-sensing domains of the prototypical eukaryotic calcium channel CaV1.1. Structure models of CaV1.1 in the activated and resting states show that VSD I and VSD IV differ greatly regarding ion-pair interactions formed between gating charges and counter-charges in the outer negative cluster. These structural differences are consistent with the specific contributions of VSD I and IV to kinetics and voltage-dependence of CaV1.1 activation, respectively. The functional relevance of the predicted interactions was confirmed by site-directed mutagenesis and patch clamp analysis. The data show that ion-pairs which stabilize the activated state cause a left-shift of the voltage-dependence of activation in VSD I and IV. In contrast, ion-pairs stabilizing resting states in VSD I slow down transition times between states and thus determine slow activation kinetics of CaV1.1. Thus, MD simulations reliably predict functionally relevant intramolecular interactions and distinct thermodynamic properties of states and different kinetics of state transitions explain the specific contributions of VSD I and IV to gating properties of CaV1.1.