Voltage-dependent Transitions Research Articles

Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.

Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a "hot" receptor, reflecting the temperature dependence of the voltage sensor's last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a "cold" receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a "loose" or "tight" conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

Voltage-dependent conformational changes of Kv1.3 channels activate cell proliferation.

The voltage-dependent potassium channel Kv1.3 has been implicated in proliferation in many cell types, based on the observation that Kv1.3 blockers inhibited proliferation. By modulating membrane potential, cell volume, and/or Ca2+ influx, K+ channels caninfluence cell cycle progression. Also, noncanonical channel functions could contribute to modulate cell proliferation independentof K+ efflux. The specificity of the requirement of Kv1.3 channels for proliferation suggests the involvement of molecule-specific interactions, but the underlying mechanisms are poorly identified. Heterologous expression of Kv1.3 channels in HEK cells has been shown to increase proliferation independently ofK+ fluxes. Likewise, some of the molecular determinants of Kv1.3-induced proliferation have been located in the C-terminus region, where individual point mutations of putative phosphorylation sites (Y447A and S459A) abolished Kv1.3-induced proliferation. Here,we investigated the mechanisms linking Kv1.3 channels to proliferation exploring the correlation between Kv1.3 voltage-dependent molecular dynamics and cell cycle progression. Using transfected HEK cells, we analyzed both the effect of changes in resting membrane potential on Kv1.3-induced proliferation and the effect of mutated Kv1.3 channels with altered voltage dependence of gating. We conclude that voltage-dependent transitions of Kv1.3 channels enable the activation of proliferative pathways. We also found that Kv1.3 associated with IQGAP3, a scaffold protein involved in proliferation, and that membrane depolarization facilitates their interaction. The functional contribution of Kv1.3-IQGAP3 interplay to cell proliferation was demonstrated both in HEK cells and in vascular smooth muscle cells. Our data indicate that voltage-dependent conformational changes of Kv1.3 are an essential element in Kv1.3-induced proliferation.

Structure and physiological function of the human KCNQ1 channel voltage sensor intermediate state.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

Temperature Sensitivity in a Potassium Channel from Land Plants

The perception of temperature is a crucial sensory mechanism of living organisms and affects cellular and metabolic processes as well as behavior. In animals, changes in environmental temperature are detected by sensory neurons that express temperature sensitive ion channels. Among these, the family of transient receptor potential (TRP) channels plays a major role. In land plants no homologues for TRP channels are found and temperature sensitive ion channels have yet to be uncovered. KAT1 is a hyperpolarization activated potassium channel from Arabidopsis thaliana that regulates stomatal opening. This channel has voltage sensing domain (VSD) and pore domain (PD) similar to depolarization activated potassium channels. KAT1 was expressed in Xenopus laevis oocytes and the cut open voltage clamp technique was used to record the hyperpolarization induced currents. Rapid changes in temperature where achieved as described in the abstract “Probing Ion Channel Thermodynamics With Temperature Jumps In Oocytes”. Dramatic changes in the kinetics of activation and deactivation where observed. Applying a temperature jump before a hyperpolarizing voltage greatly speeds up channel activation with a significant reduction in the delay of the current onset. Changing the bath temperature greatly affects the kinetics of the channel but does not affect the relative conductance versus voltage relation indicating that the voltage dependent transitions are not involved in temperature sensitivity. The activation rate can be well described by two exponentials, each one with a Q10 of about 4. These results indicate that temperature activates KAT1 channels most likely by increasing the open probability trough changes in the coupling between the VSD and PD. The proposed mechanism is different from the allosteric regulation proposed for the TRP family channels. These results provide clues into the evolution of temperature sensitivity in plants. (Support: NIH GM030376)

Outer hair cell electromotility is low-pass filtered relative to the molecular conformational changes that produce nonlinear capacitance.

The outer hair cell (OHC) of the organ of Corti underlies a process that enhances hearing, termed cochlear amplification. The cell possesses a unique voltage-sensing protein, prestin, that changes conformation to cause cell length changes, a process termed electromotility (eM). The prestin voltage sensor generates a capacitance that is both voltage- and frequency-dependent, peaking at a characteristic membrane voltage (Vh), which can be greater than the linear capacitance of the OHC. Accordingly, the OHC membrane time constant depends upon resting potential and the frequency of AC stimulation. The confounding influence of this multifarious time constant on eM frequency response has never been addressed. After correcting for this influence on the whole-cell voltage clamp time constant, we find that both guinea pig and mouse OHC eM is low pass, substantially attenuating in magnitude within the frequency bandwidth of human speech. The frequency response is slowest at Vh, with a cut-off, approximated by single Lorentzian fits within that bandwidth, near 1.5 kHz for the guinea pig OHC and near 4.3 kHz for the mouse OHC, each increasing in a U-shaped manner as holding voltage deviates from Vh Nonlinear capacitance (NLC) measurements follow this pattern, with cut-offs about double that for eM. Macro-patch experiments on OHC lateral membranes, where voltage delivery has high fidelity, confirms low pass roll-off for NLC. The U-shaped voltage dependence of the eM roll-off frequency is consistent with prestin's voltage-dependent transition rates. Modeling indicates that the disparity in frequency cut-offs between eM and NLC may be attributed to viscoelastic coupling between prestin's molecular conformations and nanoscale movements of the cell, possibly via the cytoskeleton, indicating that eM is limited by the OHC's internal environment, as well as the external environment. Our data suggest that the influence of OHC eM on cochlear amplification at higher frequencies needs reassessment.

Structural basis for activation of voltage sensor domains in an ion channel TPC1

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca2+ ions for activation.

Stochastic shielding and edge importance for Markov chains with timescale separation.

Nerve cells produce electrical impulses (“spikes”) through the coordinated opening and closing of ion channels. Markov processes with voltage-dependent transition rates capture the stochasticity of spike generation at the cost of complex, time-consuming simulations. Schmandt and Galán introduced a novel method, based on the stochastic shielding approximation, as a fast, accurate method for generating approximate sample paths with excellent first and second moment agreement to exact stochastic simulations. We previously analyzed the mathematical basis for the method’s remarkable accuracy, and showed that for models with a Gaussian noise approximation, the stationary variance of the occupancy at each vertex in the ion channel state graph could be written as a sum of distinct contributions from each edge in the graph. We extend this analysis to arbitrary discrete population models with first-order kinetics. The resulting decomposition allows us to rank the “importance” of each edge’s contribution to the variance of the current under stationary conditions. In most cases, transitions between open (conducting) and closed (non-conducting) states make the greatest contributions to the variance, but there are exceptions. In a 5-state model of the nicotinic acetylcholine receptor, at low agonist concentration, a pair of “hidden” transitions (between two closed states) makes a greater contribution to the variance than any of the open-closed transitions. We exhaustively investigate this “edge importance reversal” phenomenon in simplified 3-state models, and obtain an exact formula for the contribution of each edge to the variance of the open state. Two conditions contribute to reversals: the opening rate should be faster than all other rates in the system, and the closed state leading to the opening rate should be sparsely occupied. When edge importance reversal occurs, current fluctuations are dominated by a slow noise component arising from the hidden transitions.

Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics.

Tuning Voltage Dependent Transitions during the Extracellular Na+ Binding/Release of the Na+/K+-ATPase by External Protons

Tuning Voltage Dependent Transitions during the Extracellular Na+ Binding/Release of the Na+/K+-ATPase by External Protons

Reversible voltage dependent transition of abnormal and normal bipolar resistive switching.

Clear understanding the mechanism of resistive switching is the important prerequisite for the realization of high performance nonvolatile resistive random access memory. In this paper, binary metal oxide MoOx layer sandwiched by ITO and Pt electrodes was taken as a model system, reversible transition of abnormal and normal bipolar resistive switching (BRS) in dependence on the maximum voltage was observed. At room temperature, below a critical maximum voltage of 2.6 V, butterfly shaped I-V curves of abnormal BRS has been observed with low resistance state (LRS) to high resistance state (HRS) transition in both polarities and always LRS at zero field. Above 2.6 V, normal BRS was observed, and HRS to LRS transition happened with increasing negative voltage applied. Temperature dependent I-V measurements showed that the critical maximum voltage increased with decreasing temperature, suggesting the thermal activated motion of oxygen vacancies. Abnormal BRS has been explained by the partial compensation of electric field from the induced dipoles opposite to the applied voltage, which has been demonstrated by the clear amplitude-voltage and phase-voltage hysteresis loops observed by piezoelectric force microscopy. The normal BRS was due to the barrier modification at Pt/MoOx interface by the accumulation and depletion of oxygen vacancies.

Allostery: A lipid two-step.

A sensor of membrane depolarization controls the activity of a bound enzyme through a novel mechanism involving two sequential voltage-dependent transitions allosterically coupled to changes in the substrate specificity of the catalytic domain.

Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K(+) fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.

Voltage-Dependent Accessibility of Phosphorylation Sites at the Carboxy-Terminal Domain of Kv1.3 Channels Determines Kv1.3-Induced Cell Proliferation

The plasticity of vascular smooth muscle cells that can switch from a contractile to a proliferative phenotype associates with several cardiovascular diseases. We have previously demonstrated that changes in the expression of voltage-dependent potassium (Kv) channels associated with this proliferative phenotype. These results were validated in transfected HEK293 cells: Kv1.5 decreased proliferation and Kv1.3 increased cell proliferation independently of ion flux (Cidad et al. ATVB 2012, 32:1299-307). Our data also suggested that the movement of the voltage-sensor of Kv1.3 channels could be coupled to signaling pathways leading to proliferation.Here we explore Kv1.3 regions involved in the signaling mechanism(s) associated with proliferation, by constructing chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels without consensus phosphorylation sites. These constructs were expressed as GFP- or cherry-fusion proteins, and we studied their functional expression, their effects on proliferation and their phosphorylation state in HEK cells combining immunocytochemical, electrophysiological and EdU incorporation studies.The molecular determinants of Kv1.3-induced proliferation are located at the carboxy-terminal domain. We identified two close residues (Y447 and S459) whose mutation abolished proliferation. Moreover, proliferation was increased with chimeric Kv1.5 mutants containing this segment (YS segment) but only when located at specific place within the channel molecule. Besides the selective MEK/ERK blocker PD98059 was able to inhibit Kv1.3-induced proliferation and tyrosine phosphorylation of the channel, having no effect on Y447A mutant channel. Finally, voltage-dependent transitions of Kv1.3 from close to open conformation increased pTyr labelling.We conclude that the signaling pathway linking Kv1.3 channels with proliferation involves voltage-induced conformational changes promoting tyrosine phosphorylation. Both phosphorylation and proliferation increase by facilitating the closed to open transition, being diminished in mutant channels that reside in the inactivated state. Supported by ISCIII-FEDER RD12/0042/0006, and BFU2013-45867-R grants.

Voltage Dependent Reactions in the Non-Gastric H/K-ATPase

The non-gastric H/K-ATPase (ngHKA) is the closest relative to the Na/K-ATPase (NKA). Its catalytic subunit associates with the NKA β1 subunit to form a functional, ouabain-sensitive HKA. To study ion binding and unbinding reactions in ngHKA, we measured 86Rb uptake and ouabain-sensitive (20 mM) currents under two-electrode voltage clamp in Na+-loaded Xenopus oocytes. Although HKA transports ions in an electroneutral fashion, HKA injected oocytes presented clamp speed-limited ouabain-sensitive charge movement in response to pulses between −180 to +40 mV from −50 mV in 125 mM external N-methyl D-glucamine (NMG). The charge-voltage curve did not saturate at extreme voltages at pH 7.6, but saturated at negative voltages at pH 5.0. The charge moved by a pulse from −50 to −180 mV was 1.29 ± 0.15 nC at pH 7.6 and 0.35 ± 0.08 nC at pH 5.0. This charge was absent in uninjected oocytes (60 ± 23 pC). External K+ abolished charge movement with mildly voltage-dependent K0.5 (∼ 0.15 mM at −180 mV), while the charge moved in the presence of Na+ at pH 7.6 presented nearly identical saturation at negative voltages as NMG at pH 5.0. This demonstrates the existence of mildly voltage-dependent transitions in ngHKAs which, like the charge movement in NKA, are inhibited by the external presence of the imported ion (K+) and modified by high concentrations of the exported ions (Na+ or H+, respectively). To further address the ion binding reactions and their selectivity, we mutated K793A in the catalytic subunit to make the pump electrogenic. As expected, this mutant concomitantly displayed 86Rb uptake and Rb+-induced steady-state outward currents as well as altered transient charge movement. Unexpectedly, K793A largely reduced the pump's affinity for K+o. Supported by NSF-MCB-1243842 & NIH-NS081570-01, to PA; GM 061583 to CG

Can many subunits make light work of ion channel inactivation?

Voltage-gated K+ channels have fascinated physiologists for many decades, not just because they play important roles in a myriad of cellular functions including neurotransmission, cardiac repolarization, hormone secretion and cell volume homeostasis but also because they are exemplary nanomachines that are capable of extraordinary feats of molecular gymnastics. In recent years, the human ether a go-go related gene (hERG) K+ channel has been of particular interest as mutations in hERG result in a markedly increased risk of cardiac arrhythmias and because these channels are the molecular target for the vast majority of drugs that have had to be withdrawn from the market due to an unacceptably high risk of drug-induced cardiac arrhythmias (Vandenberg et al. 2012). Voltage-gated K+ channels transition between closed, open and inactivated states. The rates and voltage dependence of these transitions vary between family members allowing for the fine-tuning of electrical signals in different tissue types. In hERG channels, the rapid voltage dependent transition between the open and inactivated states, coupled with slow open–closed state transitions, are critical for their role in cardiac repolarization and in opposing ectopic beats (Smith et al. 1996). Despite the distinctive kinetics and voltage dependence, inactivation gating in hERG K+ channels shares many similarities with the slow ‘c-type inactivation’ in Shaker K+ channels (Smith et al. 1996). All voltage-gated K+ channels are tetrameric complexes with a single central ion conduction pathway, the extracellular end of which is lined by one selectivity filter from each of the four subunits. C-type inactivation is thought to involve rearrangements of the selectivity filter region of the channel. However, recently it has been shown that inactivation of hERG K+ channels is a significantly more complex allosteric process involving sequential rearrangements of multiple domains in the channel (Wang et al. 2011). Hitherto, it has been unclear to what extent these rearrangements occur within and/or between channel subunits. In a study published in this issue of The Journal of Physiology (Wu et al. 2014a), Wu and colleagues have utilized a subunit concatemer approach to specifically investigate whether inactivation in hERG K+ channels requires co-operative interactions between subunits. This approach allows the introduction of mutations into one or more specific subunits within the tetramer, rather than having to rely on the random association of subunits that occurs with co-expression of wild-type and mutant subunits. One issue with this approach is that three of the free N-termini and C-termini present in wild-type channels are removed in the concatemer. For channels where the N-terminus or C-terminus influences the gating transition of interest this can be a problem. Whilst deletion of the N-terminus of hERG K+ channels can influence inactivation gating (Gustina & Trudeau, 2013) previous studies have shown that concatenation of four wild-type hERG subunits did not significantly affect inactivation (Wu et al. 2014b). Initially, Wu and colleagues show that the introduction of either the S620T or the G628C/S631C mutation into a single subunit of the concatenated hERG K+ channel prevented channels from inactivating. Thus, all four subunits must be inactivation competent for the channels to inactivate. This suggests that the final, or at least a very late, step in the inactivation process requires a concerted all-or-nothing co-operative interaction between the four subunits. Interestingly, an investigation of mutants more remote from the selectivity filter showed quite distinct effects on inactivation. For example, studies of T618A (in the pore helix) and S631A (just external to the selectivity filter) suggested a sequential model of co-operative subunit interactions, whereas studies of M645C, in the pore lining S6 domain, suggested that the subunits act independently of one another during inactivation. So the answer to the question of how subunits interact during inactivation of hERG K+ channels depends very much on what stage of the process you are looking at. Previous studies of C-type inactivation in other voltage-gated K+ channels, using a similar concatemer approach, have suggested that there are co-operative interactions between subunits. However, most of these studies have only studied one or two mutations close to the selectivity filter. The study from Wu and colleagues indicates that the apparent nature of the subunit interactions is dependent on the location of the mutation studied. It would be intriguing to see if there is a similar level of variability in co-operativity for C-type inactivation in other channels. If this were the case, it would suggest that the basic process of C-type inactivation is widely conserved and that hERG K+ channel inactivation represents one variation of a common theme. The current studies add a new chapter to our understanding of the complexity of hERG K+ channel gating, but whether this level of complexity is conserved in all voltage-gated K+ channels remains to be determined.

A Molecular Voltmeter Based on Fluorescence Dynamics

In this issue, Hou et al. (1) introduce an elegant method for measuring the absolute voltage across a cell membrane with an accuracy of 10 mV. Their study presents an incisive approach to voltage imaging in which the voltage value is encoded in the temporal dynamics of fluorescence rather than in the raw fluorescence intensity. Together with an engineered microbial rhodopsin with properties amenable to the approach, the method provides a genetically encoded optical read-out independent of the reporter’s expression level and insensitive to variations in common experimental parameters. Their work presents a conceptually new avenue toward developing probes for membrane voltage useful for myriad systems from bacteria to mammalian tissue.

Opening and Closing of the HCN2 Channel Pore is Voltage-Independent

Recent studies have suggested that the opening and closing of Hyperpolarization-activated Cyclic Nucleotide-gated 'HCN' channels involve a step that is voltage-independent, which depends upon a region that resides within the S4 and S6 transmembrane domains of the channels. In the current study, we use two electrode voltage clamp of the HCN2 channel expressed in Xenopus oocytes to show that mutation of a residue site in the S6 segment, in region that likely corresponds to the pore, dramatically slows channel closing, without large effects on the rate of channel opening, and decreases the dependence of channel closing on voltage. A 6-state,but not a 4-state, cyclic allosteric model incorporating voltage-dependent transitions moving the channels between resting and active states and voltage-independent transitions between closed and open states was best able to describe the complex gating behaviour of both the wild type and pore mutant channels in response to changes in voltage. Modification of the voltage-independent closing transition recapitulates both the very slow and voltage-independent closing of mutant channel. In summary, our data supports a cyclic allosteric model of HCN2 channel gating in which the opening and closing of the pore is voltage-independent.

Multiple mechanisms underlying rectification in retinal cyclic nucleotide-gated (CNGA1) channels

In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current–voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb+ and Cs+ single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K+ channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na+ and K+.

Computational Model for Monitoring of the Membrane Potential with the Fluorescent Probe DiSC3(5)

The optical properties of cationic dicarbocyanide dyes have been shown to depend on the transmembrane electrical potential of liposomes or living cells. In this work, we developed the most complete computational model allowing optimization of experimental conditions to monitor the membrane potential of membrane vesicles composed of neutral and charged phospholipids at fixed ionic strength of incubation media in a desirable voltage range. The model allows predicting potential‐dependent changes of the fluorescent properties of DiSC3(5) on concentration of the dye and on the lipid composition of the liposomes of certain sizes. It also takes into account the values of the surface potentials of the inner and outer leaflets of the lipid bilayer, as well as their changes due to the DiSC3(5) incorporation into the lipid phase. The probe dimerization in the aqueous and lipid phases, and the possibilities of the voltage‐dependent transitions of the dimers between two leaflets of the lipid bilayer and between the aqueous and lipid phases were also included in the model, in addition to the monomer transitions. The model was adjusted to several literature data obtained with DiSC3(5) probe as an indicator of the membrane potential in experiments with liposomes and biomembrane vesicles. Colciencias (Colombia) research grants #111840820380 and #111852128625.