Syntaxin 1A inhibits regulated CFTR trafficking in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR)is an epithelial cell Cl channel, whose gating activity and membranetrafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett,D. J. Benos, M. W. Quick, and K. L. Kirk.Nature 390: 302-305, 1997), aprotein best known for its role in membrane trafficking andneurosecretion. To examine the mechanism of syntaxin 1A inhibition, weexpressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membranecapacitance and cell surface fluorescence of CFTR that contains anexternal epitope tag. cAMP stimulation elicited large increases inmembrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTRcontent. Injection of botulinum toxin/C1 rapidly reversed syntaxin'seffects on current and capacitance, indicating that they cannot beexplained by an effect on CFTR synthesis. Functional expression ofother integral membrane proteins, including Na-coupled glucosetransporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotidereceptor, and viral hemagglutinin protein, was not affected by syntaxin1A coexpression. These findings indicate that acute regulation of thenumber of CFTR Cl channels in plasma membrane is one mechanism by whichcAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTRcontent by syntaxin 1A is consistent with the concept that syntaxin andother components of the SNARE machinery are involved in regulatedtrafficking of CFTR.

     

Regulation of epithelial sodium channel activity by SARS-CoV-1 and SARS-CoV-2 proteins

Severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS-CoV-2), which causes the coronavirus disease 2019, encodes several proteins whose roles are poorly understood. We tested their ability either to directly form plasma membrane ion channels or to change functions of two mammalian plasma membrane ion channels, the epithelial sodium channel (ENaC) and the α3β4 nicotinic acetylcholine receptor. In mRNA-injected Xenopus oocytes, none of nine SARS-CoV-2 proteins or two SARS-CoV-1 proteins produced conductances, nor did co-injection of several combinations. Immunoblots for ORF8, spike (S), and envelope (E) proteins revealed that the proteins are expressed at appropriate molecular weights. In experiments on coexpression with ENaC, three tested SARS proteins (SARS-CoV-1 E, SARS-CoV-2 E, and SARS-CoV-2 S) markedly decrease ENaC currents. SARS-CoV-1 S protein decreases ENaC currents modestly. Coexpressing the E proteins but not the S proteins with α3β4 nicotinic acetylcholine receptors significantly reduces acetylcholine-induced currents. ENaC inhibition does not occur if the SARS-CoV protein mRNAs are injected 24 h after the ENaC mRNAs, suggesting that SARS-CoV proteins affect early step(s) in functional expression of channel proteins. Consistent with the hypothesis that the SARS-CoV-2 S protein-induced ENaC inhibition involves competition for available protease, mutating the furin cleavage site in SARS-CoV-2 S protein partially relieves inhibition of ENaC currents. Extending previous suggestions that SARS proteins affect ENaC currents via protein kinase C (PKC) activation, PKC activation via phorbol 12-myristate 13-acetate decreases ENaC and α3β4 activity. Phorbol 12-myristate 13-acetate application reduced membrane capacitance ～5%, presumably via increased endocytosis, but this decrease is much smaller than the SARS proteins’ effects on conductances. Also, incubating oocytes in Gö-6976, a PKCα and PKCβ inhibitor, did not alter E or S protein-induced channel inhibition. We conclude that SARS-CoV-1 and SARS-CoV-2 proteins alter the function of human plasma membrane channels, via incompletely understood mechanisms. These interactions may play a role in the coronavirus 2019 pathophysiology.     

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E_a), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E_a seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E_a and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E_a and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E_a and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Mechanosensitive ion channels

The article concentrates on the concepts of mechanosensitive ion channels that are present in practically all cells of an organism. Considered are kinetic scheme and activation principles of mechanic-sensitive ion channels. The forces affecting those channels are discussed in detail. The qualities of the channels in lipid monolayer, bilayer and real cell membrane are under consideration. Discussed are various models that analyze possibilities of channel opening depending on the membrane tension. Under discussion are the data received from studying single channels, currents in whole-cell configuration and cloned channels built into bilayer, liposomes and membrane blebs. Problems of transmitting mechanic energy to the channel through the bilayer and through the cytoskeleton are investigated. Inhibitors and activators of mechanosensitive ion channels are mentioned and their effects are considered. The functional classification of mechanosensitive ion channels is given. Described are cation SACs, potassium SACs, Ca(2+)-sensitive and Ca(2+)-insensitive SACs, anion SACs, nonselective SACs and SICs. It is proved that mechanosensitive ion channels can produce considerable currents enough to change the cell electrogenesis.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K⁺ currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K⁺ channels. The 16-pS K⁺ channels are active close to a membrane voltage of −80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K⁺ channels are active near −60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K⁺ channels were recorded at voltages positive to 0 mV. In patches containing all three types of K⁺ channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K⁺ currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K⁺ currents suggest that these single-channel K⁺ currents may underlie the macroscopic delayed K⁺ currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K⁺ currents.     

The Inhibition of Sodium Currents in Myelinated Nerve by Quaternary Derivatives of Lidocaine

The inhibition of sodium currents by quaternary derivatives of lidocaine was studied in single myelinated nerve fibers. Membrane currents were diminished little by external quaternary lidocaine (QX). QX present in the axoplasm (<0.5 mM) inhibited sodium currents by more than 90%. Inhibition occurred as the sum of a constant, tonic phase and a variable, voltage-sensitive phase. The voltage-sensitive inhibition was favored by the application of membrane potential patterns which produce large depolarizations when sodium channels are open. Voltage-sensitive inhibition could be reversed by small depolarizations which opened sodium channels. One explanation of this observation is that QX molecules enter open sodium channels from the axoplasmic side and bind within the channels. The voltage dependence of the inhibition by QX suggests that the drug binds at a site which is about halfway down the electrical gradient from inside to outside of the sodium channel.     

Membrane capacity of squid giant axon during hyper- and depolarizations

Summary The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarized and depolarized membranes. The duration of DC pulses is 20–40 msec and is long enough to permit the admittance measurements between 1 and 50 kHz. The amplitudes of DC pulses were varied between 0 and 40mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.     

4℃预处理对人中性粒细胞膜电流和极性的影响

对急性分离的人中性粒细胞采用4℃预处理是进行膜片钳实验前经常采取的步骤,但这一步骤对电生理记录结果有何影响尚无文献报道。本实验探讨这一步骤对电生理记录过程和实验结果的影响。结果显示,4℃预处理可以显著提高细胞的封接率,有利于对中性粒细胞进行电生理记录;封接率提高的原因与4℃预处理降低细胞的极性活动有关,但记录到的电压依赖性钾通道全细胞电流和大电导Ca^2＋依赖性K^＋单通道电流动力学没有显著的变化。这些结果表明,4℃预处理可能影响到细胞膜上与极性有关的脂膜变化,但对细胞膜上蛋白的功能影响较少。     

Electrogenic transport of sodium ions in cytoplasmic and extracellular ion access channels of Na+,K+-ATPase probed by admittance measurement technique

Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of the Na⁺,K⁺-ATPase have been studied by the admittance measurement technique which allows the detection of small changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. The measurements were carried out on a model system consisting of a bilayer lipid membrane, to which membrane fragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the membrane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was measured at various frequencies and in solutions with different Na⁺ concentrations. The experimentally observed frequency dependences were explained using a theoretical model assuming that Na⁺ movement through the cytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. The phosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening the extracellular access channel. The disappearance of electrogenic Na⁺ movements on the cytoplasmic side produces a negative change of capacitance and conductance, while the emergence of extracellular Na⁺ movements generates a positive change. Fitting the experimental dependences of capacitance and conductance by theoretical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the access channels. The text was submitted by the authors in English.     

Modification of sodium and potassium channel gating kinetics by ether and halothane

The effect of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rates of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out. Ether did not affect the capacitance of the axon membrane.     

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.     

Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore

Chloride intracellular channels (CLICs) are putative pore-forming glutathione-S-transferase homologs that are thought to insert into cell membranes directly from the cytosol. We incorporated soluble, recombinant human CLIC1 into planar lipid bilayers to investigate the associated ion channels, and noted that channel assembly (unlike membrane insertion) required a specific lipid mixture. The channels formed by reduced CLIC1 were similar to those previously recorded from cells and "tip-dip" bilayers, and specific anti-CLIC1 antibodies inhibited them. However, the amplitudes of the filtered single-channel currents were strictly regulated by the redox potential on the "extracellular" (or "luminal") side of the membrane, with minimal currents under strongly oxidizing conditions. We carried out covalent functional modification and site-directed mutagenesis of this controversial ion channel to test the idea that cysteine 24 is a critical redox-sensitive residue located on the extracellular (or luminal) side of membrane CLIC1 subunits, in a cysteine-proline motif close to the putative channel pore. Our findings support a simple structural hypothesis to explain how CLIC1 oligomers form pores in membranes, and suggest that native channels may be regulated by a novel mechanism involving the formation and reduction of intersubunit disulphide bonds.     

Nonselective voltage-gated ionic channels in type II taste cells

In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K⁺-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells.     

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10⁻⁶ M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10⁻⁶ M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Sodium Chloride Compartmentation in Leaf Vacuoles of the Halophyte Suaeda maritima (L.) Dum. and its Relation to Tonoplast Permeability

Single channel properties, whole vacuole currents and protonpumping capacity were investigated in the intact vacuoles andmembrane patches of leaf tonoplast from the halophyte Suaedamaritima. ATP-dependent proton pumping capacity was similarto non-halophytes whether the plants were or were not grownwith added sodium chloride (200 mM). The most abundant ion channelwas inward rectifying and had a single channel conductance of58 pS in symmetrical KCl solutions (100 mM) to 170 pS usingphysiological conditions (50/150 mM KCl/NaCl cytoplasmic side,50/450 mM KCl/NaCl vacuolar side). The channel showed all thecharacteristics of the SV type channel described in many otherspecies. In the open state these channels caused tonoplast conductancesin excess of 0.5 nS m²– but conductances were much lowerusing physiological ion concentrations and membrane potentials.In spite of the poor selectivity and the potentially large tonoplastconductance it is calculated that compartmentation of NaCl inleaf vacuoles can be sustained by about 30% of ATP-dependentproton pumping capacity. The results do not indicate any specialadaptation of the tonoplast ion channels in the halophyte. Key words: Ion-channels, patch-clamp, salt-tolerance, vacuole     

Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP

A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.