Effects of glutaraldehyde on sodium channel activation and inactivation in frog nerve fiber

The effects of glutaraldehyde on sodium channel gating were investigated in the membrane of the node of Ranvier in frog nerve fiber. It was found that treating the membrane with glutaraldehyde slows the rate of inactivation, renders the inactivation curve considerably less steep, and leads to the appearance of a steady-state current component. It also decelerated the activation rate and reduced the slope of the central portion of the activation curve, which was shifted over to depolarization at the membrane. This produced no significant change in the effective charge in the effective charge of activation as determined from the limiting logarithmic slope of the activation curve. The mechanisms possibly underlying these changes in sodium channel gating are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 579–586, September–October, 1986.     

Potential-dependent interaction of chemical reagents with the gating mechanism of the nerve fiber sodium channel

The action of two water-soluble carbodiimides and of Woodward's reagent K on the properties of the gating mechanism of sodium channels of the Ranvier node membrane was investigated. Treatment with carbodiimide solution (pH 4.8–5.2) at a potential of –80 to –100 mV was shown to delay activation and inactivation of the channels considerably and to reduce the sensitivity of both gating functions to changes in membrane potential. The effective activation charge, determined relative to the limiting logarithmic slope of the activation curve (z_ef) was reduced by 1.7 times. Treatment of the membrane under the same conditions, but at zero holding potential, induced much smaller changes in properties of the gating mechanism; under these conditions z_ef remained unchanged. Woodward's reagent at high negative potential induced the same changes in the gating system as carbodiimide at 0 mV. The action of Woodward's reagent also depended on potential, but by a lesser degree than when carbodiimides were used. The results suggest that two types of carboxyl groups exist on the outer surface of the membrane: "mobile," which perform the role of gating particles and which are moved from the surface when the channel changes into the open or inactivated state, and "immobile," which face the external solution whatever the state of the channel.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 577–590, September–October, 1984.     

A Model of sodium channel-inactivation based upon the modulated blocker

An attempt is made to model sodium channel inactivation based upon real physical processes. The principle involved, which is supported by calculation and by direct appeal to experimental results, is that the gating dipole reversal or gating charge transfer that occurs when the channel is activated, markedly modulates the electrical properties of charged groups at the channel ends. Four examples of possible mechanisms that lead to channel inactivation are described. The simple four-state model that results is able to predict: (a) the steep voltage dependence of the equilibrium inactivation characteristic without the presence of any appreciable displacement current associated with inactivation; (b) the negative shift in membrane voltage of the equilibrium inactivation characteristic relative to the activation characteristic; (c) the bell-shaped dependence of inactivation time constant on membrane voltage; (d) the similarity of the membrane voltage dependence of the time constant of recovery from inactivation, to that of inactivation itself. A brief discussion of a model for sodium channel activation based upon the same physical principle is included.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

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.     

Effects of water-soluble carbodiimide on "fast" sodium channel gating in the rat sensory neuron membrane

Currents through "fast" (tetrodotoxin-sensitive) channels before and after external application of solutions containing 100 mM 1-ethyl-3-dimethylaminopropyl)carbodiimide-HCl (WSC) were measured during volage clamping at the membrane of dialyzed neurons of rat spinal ganglia. Treating the membrane with WSC (pH 4.8–4.9) led to a 5 to 20-fold reduction in sodium conductance, a 1.5–2.5-fold deceleration in the dynamics of current increase, and less abrupt voltage-dependent sodium channel activation curves. The shifted effective charge of activation was normally halved. The WSC produced no effect on activation parameters at normal pH (7.6). It was deduced that the changes observed resulted from WSC reacting with carboxyl groups located on the outer surface of the membrane. These groups are thought to be involved in the system of charge movements of the sodium channel gating mechanism.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 46–53, January–February, 1987.     

Non-temperature related effects of osmotic pressure on inward sodium current in the membrane of isolated rat spinal ganglia neurons

The effects of osmotic pressure on inward sodium current during a change in temperature were investigated during experiments on isolated rat spinal ganglia neurons using techniques of intracellular perfusion and voltage clamping. It was found that the effect of osmotic pressure on the kinetic parameters of sodium current does not depend on temperature over a wide range of 8–40°C; the apparent values of activation energies for the activation and inactivation processes do not dependent on degree of osmolality. Overall findings would appear to indicate that the osmotic pressure effect is actually initiated by association with aqueous transmembrane flux. Opinions are expressed as to the location of the structures through which this aqueous flux passes and of the sodium channel gating mechanism, together which the molecular mechanisms of interaction between these two elements.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 518–525, July–August, 1986.     

Role of Arginine Residues on the S4 Segment of the <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Na<Superscript>+</Superscript> Channel in Voltage-sensing

The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1–S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na⁺ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-V) relationships steady-state inactivation (h _∞) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h _∞ curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.This revised version was published online in June 2005 with a corrected cover date.     

The early phase of sodium channel gating current in the squid giant axon

A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 µs. The charge displaced accounts for about 10% (or 2 nC/cm²) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above + 20 mV and below – 100 mV This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e^– and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.     

Computing transient gating charge movement of voltage-dependent ion channels

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q–V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q–V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Gating current harmonics. IV. Dynamic properties of secondary activation kinetics in sodium channel gating.

The kinetics for sodium channel gating appear to involve three coupled processes: (a) "primary" activation, (b) "secondary" activation, and (c) inactivation. Gating current data obtained in dynamic steady states with sinusoidal voltage-clamp were analyzed to give further details about the secondary activation process in sodium channel gating. Unlike primary activation and inactivation, the secondary activation kinetics involve physical processes that become defined when the data are analyzed as a function of the sinusoid frequency in addition to mean membrane potential. The effects of these processes are described, and a physical interpretation is presented.     

Activation and inactivation of batrachotoxin-modified sodium channels in the frog nerve fiber membrane

Currents through batrachotoxin (BTX)-modified sodium channels were measured under voltage clamp conditions on the Ranvier node membrane. Potential-dependence of the fraction of activated BTX-modified channels was determined on the basis of data showing nonlinearity of the momentary current-voltage characteristic curve in the region of high negative potentials. BTX induces a shift of the sodium channel activation curve toward negative potentials on average by 67 mV, but does not, under these circumstances, alter the potential-sensitivity of their activation mechanism. The results of experiments with preliminary depolarization, of varied amplitude and duration, showed that BTX-modified sodium channels are capable of partial inactivation. The high level of steady-state conduction of the modified channels is evidently due to the fact that as a result of modification by BTX the open state of the channel becomes energetically more advantageous than the inactivated state. It is concluded that the action of BTX on inactivation differs in principle from the action of pronase.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 1, pp. 18–26. January–February, 1984.     

Human PIEZO1: Removing Inactivation

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it''s noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

蝎毒耐热蛋白对大鼠海马神经元钠通道的抑制作用

应用全细胞膜片钳技术观察蝎毒耐热蛋白（scorpion venom heat resistant protein,SVHRP）对急性分离大鼠海马神经元电压依赖性钠通道的影响。结果表明,急性分离大鼠海马神经元产生的河豚毒素（tetrodotoxin,TTX）敏感的电压依赖性钠电流被SVHRP浓度依赖性地抑制,半数抑制浓度为（0．0034±0．0004）μg／mL,Hill常数为0．4361±0．0318;SVHRP可使钠通道稳态激活曲线向电压的正方向移动,正常TTX敏感的钠通道的半数激活电压（V1/2）为（-34．38±0．62）mV（n=16）,给予0．1μg／mL的SVHRP后V1/2为（-23．96±0．41）mV（n=8,P〈0．05）,斜坡因子（κ）由正常的4．52±0．52变为3．73±0．08（n=8,P〈0．05）。SVHRP亦能改变电压依赖性钠通道的稳态失活曲线,使其向电位的负方向移动,SVHRP处理前钠通道半数失活电压（V1/2）为（-32．60±1．52）mV,κ为6．73±0．51（n=16）;0．1μg／mL的SVHRP处理后V1/2变为（-50．69±2．55）mV（n=8,P〈0．01）,κ为5．49±0．72（n=8,P〈0．05）。结果提示,SVHRP能抑制电压依赖性钠电流,改变钠通道的动力学特性,抑制其激活,促进其失活,从而影响神经元的兴奋性,这可能是其抗癫痫的机制之一。