Gating Charge Research Articles

ELUCIDATING THE DIFFERENTIAL IMPACTS OF EQUIVALENT GATING-CHARGE MUTATIONS IN VOLTAGE-GATED SODIUM CHANNELS.

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 μs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure-function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

2D Carbonaceous Materials for Molecular Transport and Functional Interfaces: Simulations and Insights.

ConspectusCarbon-based two-dimensional (2D) functional materials exhibit potential across a wide spectrum of applications from chemical separations to catalysis and energy storage and conversion. In this Account, we focus on recent advances in the manipulation of 2D carbonaceous materials and their composites through computational design and simulations to address how the precise control over material structure at the atomic level correlates with enhanced functional properties such as gas permeation, selectivity, membrane transport, and charge storage. We highlight several key concepts in the computational design and tuning of 2D structures, such as controlled stacking, ion gating, interlayer pillaring, and heterostructure charge transfer.The process of creating and adjusting pores within graphene sheets is vital for effective molecular separation. Simulations show the power of controlling the offset distance between layers of porous graphene in precisely regulating the pore size to enhance gas separation and entropic selectivity. This strategy of controlled stacking extends beyond graphene to include covalent organic frameworks (COFs) such as covalent triazine frameworks (CTFs). Experimental assembly of the layers has been achieved through electrostatic interactions, thermal transformation, and control of side chain interactions.Graphene can interface with ionic liquids in various forms to enhance its functionality. A computational proof-of-concept showcases an ion-gating concept in which the interaction of anions with the pores in graphene allows the anions to dynamically gate the pores for selective gas transport. Realization of the concept has been achieved in both porous graphene and carbon molecular sieve membranes. Ionic liquids can also intercalate between graphene layers to form interlayer pillaring structures, opening the slit space. Grand canonical Monte Carlo simulations show that these structures can be used for efficient gas capture and separation. Experiments have demonstrated that the interlayer space can be tuned by the density of the pillars and that, when fully filled with ionic liquids and forming a confined interface structure, the graphene oxide membrane achieves much higher selectivity for gas separations. Moreover, graphene can interface with other 2D materials to form heterostructures where interfacial charge transfers take place and impact the function. Both ion transport and charge storage are influenced by both the local electric field and chemical interactions.Fullerene can be used as a building block and covalently linked together to construct a new type of 2D carbon material beyond a one-atom-thin layer that also has long-range-ordered subnanometer pores. The interstitial sites among fullerenes form funnel-shaped pores of 2.0-3.3 Å depending on the crystalline phase. The quasi-tetragonal phases are shown by molecular dynamics simulations to be efficient for H2 separation. In addition, defects such as fullerene vacancies can be introduced to create larger pores for the separation of organic solvents.In conclusion, the key to imputing functions to 2D carbonaceous materials is to create new interactions and interfaces and to go beyond a single-atom layer. First-principles and molecular simulations can further guide the discovery of new 2D carbonaceous materials and interfaces and provide atomistic insights into their functions.

Structural basis for hyperpolarization-dependent opening of human HCN1 channel

Hyperpolarization and cyclic nucleotide (HCN) activated ion channels are critical for the automaticity of action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN and related plant ion channels activate upon membrane hyperpolarization. Although functional studies have identified residues in the interface between the voltage-sensing and pore domain as crucial for inverted electromechanical coupling, the structural mechanisms for this unusual voltage-dependence remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 corresponding to Closed, Open, and a putative Intermediate state. Our structures reveal that the downward motion of the gating charges past the charge transfer center is accompanied by concomitant unwinding of the inner end of the S4 and S5 helices, disrupting the tight gating interface observed in the Closed state structure. This helix-coil transition at the intracellular gating interface accompanies a concerted iris-like dilation of the pore helices and underlies the reversed voltage dependence of HCN channels.

Effective suppression of Ih and INa caused by capsazepine, known to be a blocker of TRPV1 receptor

A synthetic inhibitor of capsaicin-induced TRPV1 channel activation is called capsazepine (CPZ). In this study, we aimed to explore the effects of CPZ on hyperpolarization-activated cationic current (Ih) and voltage-gated Na + current (INa) in pituitary tumor (GH3) cells. Through patch-clamp recordings, we found that CPZ concentration-dependently inhibited Ih amplitude and slowed its activation time course. The IC50 and KD values were 3.1 and 3.16 μM, respectively. CPZ also shifted the steady-state activation curve of Ih towards a more hyperpolarized potential. However, there was no change in the gating charge of the curve. A modified Markovian model predicted the CPZ-induced decrease in the voltage-dependent hysteresis of Ih. CPZ suppressed INa in GH3 cells, without altering its activation or inactivation time course. Additionally, exposure to CPZ reduced spontaneous firing. These findings suggest that CPZ’s inhibitory effects on Ih and INa are direct and not dependent on vanilloid receptor binding. This could provide light on an unidentified ionic mechanism influencing the membrane excitability of neurons and endocrine or neuroendocrine cells in vivo.

Structural basis of human Nav1.5 gating mechanisms.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

Pathogenic gating pore current conducted by autism-related mutations in the NaV1.2 brain sodium channel

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social and communication deficits and repetitive behaviors. The genetic heterogeneity of ASD presents a challenge to the development of an effective treatment targeting the underlying molecular defects. ASD gating charge mutations in the KCNQ/KV7 potassium channel cause gating pore currents (Igp) and impair action potential (AP) firing of dopaminergic neurons in brain slices. Here, we investigated ASD gating charge mutations of the voltage-gated SCN2A/NaV1.2 brain sodium channel, which ranked high among the ion channel genes with mutations in individuals with ASD. Our results show that ASD mutations in the gating charges R2 in Domain-II (R853Q), and R1 (R1626Q) and R2 (R1629H) in Domain-IV of NaV1.2 caused Igp in the resting state of ~0.1% of the amplitude of central pore current. The R1626Q mutant also caused significant changes in the voltage dependence of fast inactivation, and the R1629H mutant conducted proton-selective Igp. These potentially pathogenic Igp were exacerbated by the absence of the extracellular Mg2+ and Ca2+. In silico simulation of the effects of these mutations in a conductance-based single-compartment cortical neuron model suggests that the inward Igp reduces the time to peak for the first AP in a train, increases AP rates during a train of stimuli, and reduces the interstimulus interval between consecutive APs, consistent with increased neural excitability and altered input/output relationships. Understanding this common pathophysiological mechanism among different voltage-gated ion channels at the circuit level will give insights into the underlying mechanisms of ASD.

A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4–S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4–S5 linker and the DIII–IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.

A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.

Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel’s voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Elucidating the differential variant effects of gating charges in NaV channels

Elucidating the differential variant effects of gating charges in NaV channels

Membrane potential accelerates sugar uptake by stabilizing the outward facing conformation of the Na/glucose symporter vSGLT

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Inhibitory Perturbations of Fluvastatin on Afterhyperpolarization Current, Erg-mediated K+ Current, and Hyperpolarization-activated Cation Current in Both Pituitary GH3 Cells and Primary Embryonic Mouse Cortical Neurons.

Fluvastatin (FLV), the first synthetically derived 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is a potent inhibitor of cholesterol biosynthesis. While its primary mechanism of action is to reduce cholesterol levels, there is some evidence suggesting that it may also have effects on K+ channels. However, the overall effects of fluvastatin on ionic currents are not yet well understood. The whole-cell clamp recordings were applied to evaluate the ionic currents and action potentials of cells. Here, we have demonstrated that FLV can effectively inhibit the amplitude of erg-mediated K+ current (IK(erg)) in pituitary tumor (GH3) cells, with an IC50 of approximately 3.2µM. In the presence of FLV, the midpoint in the activation curve of IK(erg) was distinctly shifted to a less negative potential by 10mV, with minimal modification of the gating charge. However, the magnitude of hyperpolarization-activated cation current (Ih) elicited by long-lasting membrane hyperpolarization was progressively decreased, with an IC50 value of 8.7µM, upon exposure to FLV. More interestingly, we also found that FLV (5µM) could regulate the action potential and afterhyperpolarization properties in primary embryonic mouse cortical neurons. Our study presents compelling evidence indicating that FLV has the potential to impact both the amplitude and gating of the ion channels IK(erg) and Ih. We also provide credible evidence suggesting that this drug has the potential to modify the properties of action potentials and the afterhyperpolarization current in electrically excitable cells. However, the assumption that these findings translate to similar in-vivo results remains unclear.

Structural basis for severe pain caused by mutations in the voltage sensors of sodium channel NaV1.7.

Voltage-gated sodium channels in peripheral nerves conduct nociceptive signals from nerve endings to the spinal cord. Mutations in voltage-gated sodium channel NaV1.7 are responsible for a number of severe inherited pain syndromes, including inherited erythromelalgia (IEM). Here, we describe the negative shifts in the voltage dependence of activation in the bacterial sodium channel NaVAb as a result of the incorporation of four different IEM mutations in the voltage sensor, which recapitulate the gain-of-function effects observed with these mutations in human NaV1.7. Crystal structures of NaVAb with these IEM mutations revealed that a mutation in the S1 segment of the voltage sensor facilitated the outward movement of S4 gating charges by widening the pathway for gating charge translocation. In contrast, mutations in the S4 segments modified hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids that would enhance the outward movement of the gating charges. These results provide key structural insights into the mechanisms by which these IEM mutations in the voltage sensors can facilitate outward movements of the gating charges in the S4 segment and cause hyperexcitability and severe pain in IEM. Our work gives new insights into IEM pathogenesis at the near-atomic level and provides a molecular model for mutation-specific therapy of this debilitating disease.

Cell-cycle arrest at the G1/S boundary enhances transient voltage-gated ion channel expression in human and insect cells

Heterologous expression of recombinant ion channel subunits in cell lines is often limited by the presence of a low number of channels at the cell surface level. Here, we introduce a combination of two techniques: viral expression using the baculovirus system plus cell-cycle arrest at the G1/S boundary using either thymidine or hydroxyurea. This method achieved a manifold increase in the peak current density of expressed ion channels compared with the classical liposome-mediated transfection methods. The enhanced ionic current was accompanied by an increase in the density of gating charges, confirming that the increased yield of protein and ionic current reflects the functional localization of channels in the plasma membrane. This modified method of viral expression coordinated with the cell cycle arrest will pave the way to better decipher the structure and function of ion channels and their association with ion channelopathies.

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Fifty years of gating currents and channel gating.

We celebrate this year the 50th anniversary of the first electrophysiological recordings of the gating currents from voltage-dependent ion channels done in 1973. This retrospective tries to illustrate the context knowledge on channel gating and the impact gating-current recording had then, and how it continued to clarify concepts, elaborate new ideas, and steer the scientific debate in these 50 years. The notion of gating particles and gating currents was first put forward by Hodgkin and Huxley in 1952 as a necessary assumption for interpreting the voltage dependence of the Na and K conductances of the action potential. 20 years later, gating currents were actually recorded, and over the following decades have represented the most direct means of tracing the movement of the gating charges and gaining insights into the mechanisms of channel gating. Most work in the early years was focused on the gating currents from the Na and K channels as found in the squid giant axon. With channel cloning and expression on heterologous systems, other channels as well as voltage-dependent enzymes were investigated. Other approaches were also introduced (cysteine mutagenesis and labeling, site-directed fluorometry, cryo-EM crystallography, and molecular dynamics [MD] modeling) to provide an integrated and coherent view of voltage-dependent gating in biological macromolecules. The layout of this retrospective reflects the past 50 years of investigations on gating currents, first addressing studies done on Na and K channels and then on other voltage-gated channels and non-channel structures. The review closes with a brief overview of how the gating-charge/voltage-sensor movements are translated into pore opening and the pathologies associated with mutations targeting the structures involved with the gating currents.

Interplay between VSD, pore and membrane lipids in electromechanical coupling in HCN channels.

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a 'domino effect' during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

Domain- and state-specific shape of the electric field tunes voltage sensing in voltage-gated sodium channels

The ability to sense transmembrane voltage underlies most physiological roles of voltage-gated sodium (Nav) channels. Whereas the key role of their voltage-sensing domains (VSDs) in channel activation is well established, the molecular underpinnings of voltage coupling remain incompletely understood. Voltage-dependent energetics of the activation process can be described in terms of the gating charge that is defined by coupling of charged residues to the external electric field. The shape of the electric field within VSDs is therefore crucial for the activation of voltage-gated ion channels. Here, we employed molecular dynamics simulations of cardiac Nav1.5 and bacterial NavAb, together with our recently developed tool g_elpot, to gain insights into the voltage-sensing mechanisms of Nav channels via high-resolution quantification of VSD electrostatics. In contrast to earlier low-resolution studies, we found that the electric field within VSDs of Nav channels has a complex isoform- and domain-specific shape, which prominently depends on the activation state of a VSD. Different VSDs vary not only in the length of the region where the electric field is focused but also differ in their overall electrostatics, with possible implications in the diverse ion selectivity of their gating pores. Due to state-dependent field reshaping, not only translocated basic but also relatively immobile acidic residues contribute significantly to the gating charge. In the case of NavAb, we found that the transition between structurally resolved activated and resting states results in a gating charge of 8e, which is noticeably lower than experimental estimates. Based on the analysis of VSD electrostatics in the two activation states, we propose that the VSD likely adopts a deeper resting state upon hyperpolarization. In conclusion, our results provide an atomic-level description of the gating charge, demonstrate diversity in VSD electrostatics, and reveal the importance of electric-field reshaping for voltage sensing in Nav channels.

Characterization of two pathological gating-charge substitutions in Cav1.4 L-type calcium channels

ABSTRACT Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.