Gating Properties Research Articles

Structural determinants of voltage-gating properties in calcium channels.

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

An integrated Janus porous membrane with controllable under-oil directional water transport and fluid gating property for oil/water emulsion separation

Directional liquid transport driven by the interface or surface property of Janus porous membrane owes great significance to many applications such as separation technology, fog collection, and energy devices. The Janus porous membranes have successfully demonstrated an efficient oil self-transport in water and also enable directional water transport in air or oil-water interface. However, the directional water transport in oil environment is a challenge because of the relatively low surface tension of oil. In this work, a novel integrated Janus porous membrane is developed for the first time to construct self-propelled energy free directional water transport in oil system that successfully demonstrated to be a facile “water diode” for under-oil water one-way regulation. Additionally, this Janus porous membrane exhibits an interesting tunable fluid-gating behavior for either water or oil, using optimized conditions. The current findings may promote the innovation towards directional liquid transport science and will open up new avenues for diverse on-demand liquids manipulation scenarios such as water-in-heavy oil emulsion separation.

Voltage-Gated Ca2+ Channels in Dopaminergic Substantia Nigra Neurons: Therapeutic Targets for Neuroprotection in Parkinson's Disease?

The loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SN) underlies the core motor symptoms of the progressive movement disorder Parkinson's disease (PD). To date, no treatment to prevent or slow SN DA neurodegeneration exists; thus, the identification of the underlying factors contributing to the high vulnerability of these neurons represents the basis for the development of novel therapies. Disrupted Ca2+ homeostasis and mitochondrial dysfunction seem to be key players in the pathophysiology of PD. The autonomous pacemaker activity of SN DA neurons, in combination with low cytosolic Ca2+ buffering, leads to large somatodendritic fluctuations of intracellular Ca2+ levels that are linked to elevated mitochondrial oxidant stress. L-type voltage-gated Ca2+ channels (LTCCs) contribute to these Ca2+ oscillations in dendrites, and LTCC inhibition was beneficial in cellular and in vivo animal models of PD. However, in a recently completed phase 3 clinical trial, the dihydropyridine (DHP) LTCC inhibitor isradipine failed to slow disease progression in early PD patients, questioning the feasibility of DHPs for PD therapy. Novel evidence also suggests that R- and T-type Ca2+ channels (RTCCs and TTCCs, respectively) represent potential PD drug targets. This short review aims to (re)evaluate the therapeutic potential of LTCC, RTCC, and TTCC inhibition in light of novel preclinical and clinical data and the feasibility of available Ca2+ channel blockers to modify PD disease progression. I also summarize their cell-specific roles for SN DA neuron function and describe how their gating properties allow activity (and thus their contribution to stressful Ca2+ oscillations) during pacemaking.

The de novo CACNA1A pathogenic variant Y1384C associated with hemiplegic migraine, early onset cerebellar atrophy and developmental delay leads to a loss of Cav2.1 channel function

CACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.

Modeling the Crosstalk between Ryanodine and IP3 Receptors in Cardiac Myocytes

Ryanodine receptors (RYR) and IP3 receptors (IP3R) are calcium channels expressed on the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane in various cell types. Both the spatial localization and the distinct gating properties of these channels contribute to the diverse cellular functions controlled by intracellular calcium signaling. It is known that both RYRs and IP3Rs are expressed on the SR membrane of cardiac myocytes and that the expression of IP3Rs on the SR at dyadic junctions is increased in cardiac disease, and evidence that calcium release through IP3Rs can influence RyR-mediate calcium release has been described. However, despite the suggested functional role for crosstalk between RYRs and IP3Rs, especially in cardiac disease, most previous mathematical models of myocyte calcium signaling have accounted for only RYRs in isolation. Here, we propose a mathematical model of intracellular calcium signaling that incorporates both RYRs and IP3Rs and can be used to develop quantitative predictions of crosstalk between the two channels. This model considers the spatial arrangement of RYRs and IP3Rs relative to one another based on published co-localization studies using immunohistochemistry and simulates the stochastic opening and closing of individual receptors based on previously published models of calcium sparks and puffs. In these models, gating of RYRs depends on the local cytosolic and JSR calcium concentration as well as the number of neighboring RYRs that are open while IP3R gating depends primarily on local cytosolic calcium and IP3 concentrations. Based on preliminary results, we hypothesize that the subcellular spatial remodeling and increased expression of IP3Rs that occurs in cardiac disease will increase the probability of diastolic calcium spark events, which may contribute to the increased risk of arrhythmia in those with cardiac diseases such as hypertrophy and heart failure.

Machine Learning Techniques to Classify the Severity of Novel SCN5A Variants

Gain-of-function mutations in the SCN5A gene, which encodes the α subunit of the major cardiac Na+ channel (Nav1.5), can promote Long-QT Syndrome Type 3 (LQT3). Mutations can affect the gating properties of the channel, as well as its protein expression and trafficking. We adopted multiscale mathematical modeling and machine learning to identify which molecular properties make certain genetic variants pathological. We began with a published mathematical model describing Nav1.5 gating and employed a genetic algorithm to optimize its parameters to the Na+ current (INa) and action potential (AP) of healthy human cardiomyocytes.

Growth and thermal stability studies of layered GaTe single crystals in inert atmospheres

In this study, we investigated the thermal stability properties in inert atmospheres of GaTe single crystals fabricated by a temperature gradient technique. The obtained crystals possess a monoclinic layered structure with high crystalline quality. To explore the thermal stability of GaTe, the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were performed up to 1173 ​K in N2 and Ar atmospheres. GaTe crystals are thermally stable up to 700 ​K in Ar and stable up to 935 ​K in N2 due to the protective role of the physically adsorbed N2 molecules on GaTe surface. Annealing effect on the structural and optical properties of GaTe were examined after thermal treatment in N2 atmosphere at different temperatures; 573, 673, 773, and 873 ​K. Three prominent Raman modes at 97 ​cm−1 (Ag), 145 ​cm−1 (Ag), and 158 ​cm−1 (Bg) corresponding to the monoclinic GaTe phase almost remained, suggesting no structural damage at elevated temperatures. Especially, the photoluminescence (PL) intensity is improved as the annealing temperature increases and reaches a maximum at 673 ​K owing to the enhancement in the GaTe crystallinity. Above 873 ​K, we observed a significant reduction in the PL intensity, which is attributed to the vacancies induced by tellurium evaporation.

Reduced current density, partially rescued by mexiletine, and depolarizing shift in activation of SCN5A W374G channels as a cause of severe form of Brugada syndrome.

Background SCN5A‐related Brugada syndrome (BrS) can be caused by multiple mechanisms including trafficking defects and altered channel gating properties. Most SCN5A mutations at pore region cause trafficking defects, and some of them can be rescued by mexiletine (MEX).ObjectiveWe recently encountered symptomatic siblings with BrS and sought to identify a responsible mutation and reveal its biophysical defects.MethodsTarget panel sequencing was performed. Wild‐type (WT) or identified mutant SCN5A was transfected into tsA201 cells. After incubation of transfected cells with or without 0.1 mM MEX for 24–36 hr, whole‐cell sodium currents (INa) were recorded using patch‐clamp techniques.ResultsThe proband was 29‐year‐old male who experienced cardiopulmonary arrest. Later, his 36‐year‐old sister, who had been suffering from recurrent episodes of syncope since 12 years, was diagnosed with BrS. An SCN5A W374G mutation, located at pore region of domain 1 (D1 pore), was identified in both. The peak density of W374G‐INa was markedly reduced (WT: 521 ± 38 pA/pF, W374G: 60 ± 10 pA/pF, p < .01), and steady‐state activation (SSA) was shifted to depolarizing potentials compared with WT‐INa (V1/2‐WT: −39.1 ± 0.8 mV, W374G: −30.9 ± 1.1 mV, p < .01). Incubation of W374G‐transfected cells with MEX (W374G‐MEX) increased INa density, but it was still reduced compared with WT‐INa (W374G‐MEX: 174 ± 19 pA/pF, p < .01 versus W374G, p < .01 versus WT). The SSA of W374G‐MEX‐INa was comparable to W374G‐INa (V1/2‐W374G‐MEX: −31.6 ± 0.7 mV, P = NS).ConclusionsReduced current density, possibly due to a trafficking defect, and depolarizing shift in activation of SCN5A W374G are underlying biophysical defects in this severe form of BrS. Trafficking defects of SCN5A mutations at D1 pore may be commonly rescued by MEX.

Changes in Resurgent Sodium Current Contribute to the Hyperexcitability of Muscles in Patients with Paramyotonia Congenita.

Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Nav1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Nav1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Nav1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype.

Implementation of biomolecular logic gate using DNA and electrically doped GaAs nano-pore: a first principle paradigm.

One of the emerging areas of today's research arena is molecular modeling and molecular computing. The molecular logic gate can be theoretically implemented from single-strand DNA which consists of four basic nucleobases. In this study, the electronic transmission characteristics of DNA chain are investigated to form the logic gate. This biomolecular single-strand DNA chain is passed through an electrically doped gallium-arsenide nano-pore to achieve reasonably improved transmission along <1 1 1> direction. Current-voltage characteristic and device density of states with HOMO-LUMO plot of the device are explained along with the conductivity of the device to confirm the characteristics of some important logic gates like a universal gate. Ultimately the property of resistivity proves the law of Boolean logic of AND gate and universal logic gate, viz., NAND and NOR gate. All the electronic properties of the Boolean logic gate are explored based on the first principle approach by non-equilibrium Green's function coupled with density functional theory in room temperature.

A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

V374A KCND3 Pathogenic Variant Associated With Paroxysmal Ataxia Exacerbations.

ObjectiveAtaxia channelopathies share common features such as slow motor progression and variable degrees of cognitive dysfunction. Mutations in potassium voltage-gated channel subfamily D member 3 (KCND3), encoding the K+ channel, Kv4.3, are associated with spinocerebellar ataxia (SCA) 19, allelic with SCA22. Mutations in potassium voltage-gated channel subfamily C member 3 (KCNC3), encoding another K+ channel, Kv3.3, cause SCA13. First, a comprehensive phenotype assessment was carried out in a family with autosomal dominant ataxia harboring 2 genetic variants in KCNC3 and KCND3. To evaluate the physiological impact of these variants on channel currents, in vitro studies were performed.MethodsClinical and psychometric evaluations, neuroimaging, and genotyping of a family (mother and son) affected by ataxia were carried out. Heterozygous and homozygous Kv3.3 A671V and Kv4.3 V374A variants were evaluated in Xenopus laevis oocytes using 2-electrode voltage-clamp. The influence of Kv4 conductance on neuronal activity was investigated computationally using a Purkinje neuron model.ResultsThe main clinical findings were consistent with adult-onset ataxia with cognitive dysfunction and acetazolamide-responsive paroxysmal motor exacerbations in the index case. Despite cognitive deficits, fluorodeoxyglucose (FDG)-PET displayed hypometabolism mainly in the severely atrophic cerebellum. Genetic analyses revealed the new variant c.1121T>C (V374A) in KCND3 and c.2012T>C (A671V) in KCNC3. In vitro electrophysiology experiments on Xenopus oocytes demonstrated that the V374A mutant was nonfunctional when expressed on its own. Upon equal co-expression of wild-type (WT) and V374A channel subunits, Kv4.3 currents were significantly reduced in a dominant negative manner, without alterations of the gating properties of the channel. By contrast, Kv3.3 A671V, when expressed alone, exhibited moderately reduced currents compared with WT, with no effects on channel activation or inactivation. Immunohistochemistry demonstrated adequate cell membrane translocation of the Kv4.3 V374A variant, thus suggesting an impairment of channel function, rather than of expression. Computational modeling predicted an increased Purkinje neuron firing frequency upon reduced Kv4.3 conductance.ConclusionsOur findings suggest that Kv4.3 V374A is likely pathogenic and associated with paroxysmal ataxia exacerbations, a new trait for SCA19/22. The present FDG PET findings contrast with a previous study demonstrating widespread brain hypometabolism in SCA19/22.

Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel.

SCN5A gene encodes the voltage-gated sodium channel NaV1.5 which is composed of a pore-forming α subunit of the channel. Asparagine (N)-linked glycosylation is one of the common post-translational modifications in proteins. The aim of this study was to investigate impact of N-linked glycosylation disruption on the Na+ channel, and the mechanism by which glycosylation regulates the current density and gating properties of the Na+ channel. The NaV1.5-Na+ channel isoform (α submit) derived from human was stably expressed in human embryonic kidney (HEK)-293 cells (Nav1.5-HEK cell). We applied the whole-cell patch-clamp technique to study the impact of N-linked glycosylation disruption in Nav1.5-HEK cell. Inhibition of the N-glycosylation with tunicamycin caused a significant increase of NaV1.5 channel current (INa) when applied for 24h. Tunicamycin shifted the steady-state inactivation curve to the hyperpolarization direction, whereas the activation curve was unaffected. Recovery from inactivation was prolonged, while the fast phase (τfast) and the slow phase (τslow) of the current decay was unaffected by tunicamycin. INa was unaffected by tunicamycin in the present of a proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucy-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide], while it was significantly increased by tunicamycin in the presence of a lysosome inhibitor butyl methacrylate (BMA). These findings suggest that N-glycosylation disruption rescues the NaV1.5 channel possibly through the alteration of ubiquitin-proteasome activity, and changes gating properties of the NaV1.5 channel by modulating glycan milieu of the channel protein.

DEG/ENaC Ion Channels in the Function of the Nervous System: From Worm to Man.

DEG/ENaC channels are voltage-independent Na+/Ca2+ channels that are conserved across species and are expressed in many different cell types and tissues, where they contribute to a wide array of physiological functions from transepithelial Na+ transport, to sensory perception, and learning and memory. In this chapter, we focus on the members of this family that are expressed in the nervous system, grouping them based on their function. Structurally, DEG/ENaC channels are trimers formed by either identical or homologous subunits, each one protruding from the plasma membrane like a clenched hand. Crystallographic studies on chicken ASIC1a in the closed, inactivated, and open states revealed important details about the gating and permeation properties of these channels, and overall they show that the extracellular domain of the channel undergoes large conformational changes during gating. The vast majority of the channel's extracellular domain is conserved across different members and species; however, key changes including the insertion of extra loops near the finger and palm domains most likely confers gating specificity. Indeed, DEG/ENaC channels are gated by a wide range of stimuli, including mechanical forces, protons, and peptides, owing to the wide array of physiological functions they serve. Interestingly, DEG/ENaC channels are not only expressed in neurons but also in glia. Work in C. elegans is now beginning to shed new light on the role of glial DEG/ENaC in the function of the nervous system and suggests that they may be implicated in controlling ionic concentrations in the extracellular microenvironment. Finally, DEG/ENaC channels can become toxic and cause neuronal death when they are hyperactivated by genetic mutations or prolonged acidosis causing them to contribute to neuronal demise in stroke and ischemia. Taken together, molecular, structural, and behavioral work on DEG/ENaC channels expressed in the nervous system of different species highlights the crucial role of these channels in neuronal function. These data place DEG/ENaC channels in an excellent position for being considered as drug targets for the treatment of several neurological conditions and disorders from pain to epilepsy and ischemia.

Signatures for coexistence of monoclinic and hexagonal phases in GaTe nanoflakes

The burgeoning two-dimensional (2D) layered materials provide a powerful strategy to realize efficient light-emitting devices. Among them, gallium telluride (GaTe) nanoflakes, showing strong photoluminescence (PL) emission from multilayer to bulk crystal, relax the stringent fabrication requirements of nanodevices. However, detailed knowledge on the optical properties of GaTe varies as layer thickness is still missing. Here we perform thickness-dependent PL and Raman spectra, as well as temperature-dependent PL spectra of GaTe nanoflakes. Spectral analysis reveals a spectroscopic signature for the coexistence of both the monoclinic and hexagonal phases in GaTe nanoflakes. To understand the experimental results, we propose a crystal structure where the hexagonal phase is on the top and bottom of nanoflakes while the monoclinic phase is in the middle of the nanoflakes. On the basis of temperature-dependent PL spectra, the optical gap of the hexagonal phase is determined to be 1.849 eV, which can only survive under temperature higher than 200 K with the increasing phonon population. Furthermore, the strength of exciton-phonon interaction of the hexagonal phase is estimated to be 1.24 meV/K. Our results prove the coexistence of dual crystalline phases in multilayer GaTe nanoflakes, which may provoke further exploration of phase transformation in GaTe materials, as well as new applications in 2D light-emitting diodes and heterostructure-based optoelectronics.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary β subunits. Detailed information on the structure–function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate β subunits, the human β1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II–III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.

Dynamically Modulating Gating Process of Nanoporous Membrane at Sub-2 Nm Scale

Gating properties of nanochannels, as a key nanofluidic behavior, holds huge applications in sensing, mass transportation and separation. It has been widely studied the terminal “open” and “close” states in response to changes of environments, for prepared nanochannels. Yet to date, it remains great challenge to control the dynamic gating process. Here in this manuscript, we modulate the dynamically electric gating process in polypyrrole (PPy)-based nanoporous membranes system at sub-2 nm speed. The nano-confinement environment renders the polymer chain with excellent electrochemical property and the polymer film swells or contracts in a controlled speed accompanied by a reversible counter-ion uptake or expulsion. With this switching process, the thickness of the polymer changes by 83%, resulting a fully closed gating state. Besides, we directly observe the successive polymer chain variation at nanoscale (ca. 10 nm) by the AFM topography in situ . This small operation voltages and ultra-high strain scope, along with biocompatible materials, make this design promising for smart nanorobot and wearable applications.

Physiological Functions, Biophysical Properties, and Regulation of KCNQ1 (KV7.1) Potassium Channels.

KCNQ1 (KV7.1) K+ channels are expressed in multiple tissues, including the heart, pancreas, colon, and inner ear. The gene encoding the KCNQ1 protein was discovered by a positional cloning effort to determine the genetic basis of long QT syndrome, an inherited ventricular arrhythmia that can cause sudden death. Mutations in KCNQ1 can also cause other types of arrhythmia (i.e., short QT syndrome, atrial fibrillation) and the gene may also have a role in diabetes and certain cancers. KCNQ1 α-subunits can partner with accessory β-subunits (KCNE1-KCNE5) to form K+-selective channels that have divergent biophysical properties. In the heart, KCNQ1 α-subunits coassemble with KCNE1 β-subunits to form channels that conduct IKs, a very slowly activating delayed rectifier K+ current. KV7.1 channels are highly regulated by PIP2, calmodulin, and phosphorylation, and rich pharmacology includes blockers and gating modulators. Recent biophysical studies and a cryo-EM structure of the KCNQ1-calmodulin complex have provided new insights into KV7.1 channel function, and how interactions between KCNQ1 and KCNE subunits alter the gating properties of heteromultimeric channels.

Stabilization of negative activation voltages of Cav1.3 L-Type Ca2+-channels by alternative splicing

ABSTRACT -->Low voltage-activated Cav1.3 L-type Ca2+-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3S to activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3S further shifts activation (-3 to -4 mV) and inactivation (-4 to -6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than -12 mV. In contrast, we found no evidence for gating changes of the CACNA1D missense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome. Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

Effect of Dielectric Fabrication Techniques on Graphene Gating

One of the exciting features of graphene is a possibility to affect its electrical, optical, and chemical properties by gating, that is, by application of an electric field. This requires reasonably large fields (at the level of 1 V/nm necessary to induce relevant electron density changes) applied over a gating dielectric material. At these fields, most dielectrics show some conduction, which leads to an important question: what is the best dielectric to gate graphene? Here, we show that this question is imprecise as a dielectric material produced by different fabrication methods can exhibit dramatically different gating properties. Namely, we show that two oxide dielectrics (hafnia and alumina) result in positive hysteresis of graphene gating characteristics being fabricated by atomic layer deposition and negative hysteresis being fabricated by electron beam evaporation. We attribute this behavior to the stoichiometry of the samples and oxygen ion migration. It implies that oxide dielectrics should be avoided in graphene gated devices working at room temperatures.