Internal block of human heart sodium channels by symmetrical tetra- alkylammoniums

The human heart Na channel (hH1) was expressed by transient transfection in tsA201 cells, and we examined the block of Na current by a series of symmetrical tetra-alkylammonium cations: tetramethylammonium (TMA), tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium (TBA), and tetrapentylammonium (TPeA). Internal TEA and TBA reduce single-channel current amplitudes while having little effect on single channel open times. The reduction in current amplitude is greater at more depolarized membrane potentials. Analysis of the voltage-dependence of single-channel current block indicates that TEA, TPrA and TBA traverse a fraction of 0.39, 0.52, and 0.46 of the membrane electric field to reach their binding sites. Rank potency determined from single-channel experiments indicates that block increases with the lengths of the alkyl side chains (TBA > TPrA > TEA > TMA). Internal TMA, TEA, TPrA, and TBA also reduce whole-cell Na currents in a voltage-dependent fashion with increasing block at more depolarized voltages, consistent with each compound binding to a site at a fractional distance of 0.43 within the membrane electric field. The correspondence between the voltage dependence of the block of single-channel and macroscopic currents indicates that the blockers do not distinguish open from closed channels. In support of this idea TPrA has no effect on deactivation kinetics, and therefore does not interfere with the closing of the activation gates. At concentrations that substantially reduce Na channel currents, TMA, TEA, and TPrA do not alter the rate of macroscopic current inactivation over a wide range of voltages (-50 to +80 mV). Our data suggest that TMA, TEA, and TPrA bind to a common site deep within the pore and block ion transport by a fast-block mechanism without affecting either activation or inactivation. By contrast, internal TBA and TPeA increase the apparent rate of inactivation of macroscopic currents, suggestive of a block with slower kinetics.     

Ionic blockade of the rat connexin40 gap junction channel by large tetraalkylammonium ions.

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.     

Mechanism of asymmetric block of K channels by tetraalkylammonium ions in mouse neuroblastoma cells

Experiments were performed to compare the mechanism of block of voltage-dependent K channels by various short and long alkyl chain tetraalkylammonium (TAA) ions at internal and external sites. Current through single channels was recorded from excised membrane patches of cultured neuroblastoma cells using the patch-clamp technique. All of the TAA derivatives tested blocked the open channel when applied to either side of the membrane. Tetraethylammonium (TEA) reduced the amplitude of current through the open channel. Tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) reduced the open time as a function of the concentration. An additional nonconducting state was observed when TBA or TPeA was applied internally or externally, due to the presence of a drug-bound and blocked state of the channel. The closing rate under control conditions was similar to that in the presence of external tetramethylammonium (TMA), suggesting that channel closing is independent of external drug binding. The concentration for half maximal block of the channel by external TEA was 80 microM. The channel was less sensitive to internal TEA, which half blocked the channel at 27 mM. The dissociation rate of long alkyl chain TAA ions from the channel was slower when applied to the inside, compared to external application, suggesting the presence of distinct internal and external receptors. Long alkyl chain TAA derivatives, such as TBA had a faster association rate with the open channel when applied to the inside of the membrane than when applied to the outside.     

Block of neuronal chloride channels by tetraethylammonium ion derivatives

The block by the symmetric tetraethylammonium (TEA) ion derivatives tetrapropylammonium (TPrA), tetrabutylammonium (TBA), and tetrapentylammonium (TPeA) ions of fast chloride channels in acutely dissociated rat cortical neurons was studied with the excised inside- out configuration of the patch-clamp technique. When applied to the intracellular membrane surface, all three of the quaternary ammonium compounds (QAs) induced the appearance of short-lived closed states in a manner consistent with a blocking mechanism where the blocker preferentially binds to the open kinetic state and completely blocks ion current through the channel. The drug must leave the channel before the channel can return to a closed state. The mechanism of block was studied using one-dimensional dwell-time analysis. Kinetic models were fit to distributions of open and closed interval durations using the Q- matrix approach. The blocking rate constants for all three of the QAs were similar with values of approximately 12-20 x 10(6) M-1s-1. The unblocking rates were dependent on the size or hydrophobicity of the QA with the smallest derivative, TPrA, inducing a blocked state with a mean lifetime of approximately 90 microseconds, while the most hydrophobic derivative, TPeA, induced a blocked state with a mean lifetime of approximately 1 ms. Thus, it appears as though quaternary ammonium ion block of these chloride channels is nearly identical to the block of many potassium channels by these compounds. This suggests that there must be structural similarities in the conduction pathway between anion and cation permeable channels.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Permeation and inhibition of polycystin-L channel by monovalent organic cations

Polycystin-L (PCL), homologous to polycystin-2 (71% similarity in protein sequence), is the third member of the polycystin family of proteins. Polycystin-1 and -2 are mutated in autosomal dominant polycystic kidney disease, but the physiological role of PCL has not been determined. PCL acts as a Ca-regulated non-selective cation channel permeable to mono- and divalent cations. To further understand the biophysical and pharmacological properties of PCL, we examined a series of organic cations for permeation and inhibition, using single-channel patch clamp and whole-cell two-microelectrode voltage clamp techniques in conjunction with Xenopus oocyte expression. We found that PCL is permeable to organic amines, methlyamine (MA, 3.8 A), dimethylamine (DMA, 4.6 A) and triethylamine (TriEA, 6 A), and to tetra-alkylammonium cation (TAA) tetra-methylammonium (TMA, 5.5-6.4 A). TAA compounds tetra-ethylammonium (TEA, 6.1-8.2 A) and tetra-propylammonium (TPA, 9.8 A) were impermeable through PCL and exhibited weak inhibition on PCL (IC50 values>13 mM). Larger TAA cations tetra-butylammonium (TBA, 11.6 A) and tetra-pentylammonium (TPeA, 13.2 A) were impermeable through PCL as well and showed strong inhibition (IC50 values of 2.7 mM and 1.3 microM, respectively). Inhibition by TBA was on decreasing the single-channel current amplitude and exhibited no effect on open probability (NPo) or mean open time (MOT), suggesting that it blocks the PCL permeation pathway. In contract, TEA, TPA and TPeA reduced NPo and MOT values but had no effect on the amplitude, suggesting their binding to a different site in PCL, which affects the channel gating. Taken together, our studies revealed that PCL is permeable to organic amines and TAA cation TMA, and that inhibition of PCL by large TAA cations exhibits two different mechanisms, presumably through binding either to the pore pathway to reduce permeant flux or to another site to regulate the channel gating. These data allow to estimate a channel pore size of approximately 7 A for PCL.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker

A pair of conserved methionine residues, located on the cytoplasmic linker between segments S4 and S5 in the fourth domain of human heart Na channels (hH1), plays a role in the kinetics and voltage dependence of inactivation. Substitution of these residues by either glutamine (M1651M1652/QQ) or alanine (MM/AA) increases the inactivation time constant (tau) at depolarized voltages, shifts steady-state inactivation (h infinity) in a depolarized direction, and decreases the time constant for recovery from inactivation. The data indicate that the mutations affect the rate constants for both binding and unbinding of a hypothetical inactivation particle from its binding site. Cytoplasmic application of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W.A. Catterall. 1994. Neuron. 12: 1041-1048.). KIFMK produces a concentration-dependent, voltage-independent increase in the decay rate of MM/QQ and MM/AA currents at positive membrane potentials (Ki approximately 30 microM), while producing only a small increase in the decay rate of wild-type currents at a concentration of 200 microM. Although MM/QQ inactivates approximately 2.5-fold faster than MM/AA in the absence of peptide, the estimated rate constants for peptide block and unblock do not differ in these mutants. External Na+ ions antagonize the block by cytoplasmic KIFMK of MM/AA channels, but not the inactivation kinetics of this mutant in the absence of peptide. The effect of external [Na+] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channels when they are open and that peptide block does not mimic the inactivation process.     

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.     

Effects of gadolinium on ion channels in the myelinated axon of Xenopus laevis: four sites of action.

The action of gadolinium (Gd3+) on ion currents in myelinated axons of Xenopus laevis was investigated with the voltage clamp technique. The analysis revealed the following effects. (i) The potential-dependent parameters of both Na and K channels were shifted. The shift was equally large for activation, inactivation, and activation time constant curves (+9 mV for 100 microM Gd3+). The effects could be explained by screening of fixed surface charges at a density of -1.2 e nm-2. (ii) The rate of gating for both Na and K channels was reduced more than predicted from the shift. This effect could be quantified as a scaling (by a factor 3 and 5 respectively at 100 microM Gd3+) of the activation time constant curves. (iii) An activation- and inactivation-independent block of both Na and K channels, obeying 1:1 stoichiometry with a Kd value of about 70 microM potential-independent block of leakage current, obeying 1:2 stoichiometry with a Kd value of 600 microM. (iv) The analysis suggests separate binding sites for the effects, comprising high affinity modulatory and blocking sites on the channel proteins and low affinity receptors on phospholipids, associated with the effect on the leakage current.     

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.     

Permeation and inhibition of polycystin-l channel by monovalent organic cations

Polycystin-L (PCL), homologous to polycystin-2 (71% similarity in protein sequence), is the third member of the polycystin family of proteins. Polycystin-1 and -2 are mutated in autosomal dominant polycystic kidney disease, but the physiological role of PCL has not been determined. PCL acts as a Ca-regulated non-selective cation channel permeable to mono- and divalent cations. To further understand the biophysical and pharmacological properties of PCL, we examined a series of organic cations for permeation and inhibition, using single-channel patch clamp and whole-cell two-microelectrode voltage clamp techniques in conjunction with Xenopus oocyte expression. We found that PCL is permeable to organic amines, methlyamine (MA, 3.8 Å), dimethylamine (DMA, 4.6 Å) and triethylamine (TriEA, 6 Å), and to tetra-alkylammonium cation (TAA) tetra-methylammonium (TMA, 5.5-6.4 Å). TAA compounds tetra-ethylammonium (TEA, 6.1-8.2 Å) and tetra-propylammonium (TPA, 9.8 Å) were impermeable through PCL and exhibited weak inhibition on PCL (IC₅₀ values>13 mM). Larger TAA cations tetra-butylammonium (TBA, 11.6 Å) and tetra-pentylammonium (TPeA, 13.2 Å) were impermeable through PCL as well and showed strong inhibition (IC₅₀ values of 2.7 mM and 1.3 μM, respectively). Inhibition by TBA was on decreasing the single-channel current amplitude and exhibited no effect on open probability (NP_o) or mean open time (MOT), suggesting that it blocks the PCL permeation pathway. In contract, TEA, TPA and TPeA reduced NP_o and MOT values but had no effect on the amplitude, suggesting their binding to a different site in PCL, which affects the channel gating. Taken together, our studies revealed that PCL is permeable to organic amines and TAA cation TMA, and that inhibition of PCL by large TAA cations exhibits two different mechanisms, presumably through binding either to the pore pathway to reduce permeant flux or to another site to regulate the channel gating. These data allow to estimate a channel pore size of ∼7 Å for PCL.     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

Block of CaV1.2 channels by Gd3+ reveals preopening transitions in the selectivity filter

Using the lanthanide gadolinium (Gd(3+)) as a Ca(2+) replacing probe, we investigated the voltage dependence of pore blockage of Ca(V)1.2 channels. Gd(+3) reduces peak currents (tonic block) and accelerates decay of ionic current during depolarization (use-dependent block). Because diffusion of Gd(3+) at concentrations used (<1 microM) is much slower than activation of the channel, the tonic effect is likely to be due to the blockage that occurred in closed channels before depolarization. We found that the dose-response curves for the two blocking effects of Gd(3+) shifted in parallel for Ba(2+), Sr(2+), and Ca(2+) currents through the wild-type channel, and for Ca(2+) currents through the selectivity filter mutation EEQE that lowers the blocking potency of Gd(3+). The correlation indicates that Gd(3+) binding to the same site causes both tonic and use-dependent blocking effects. The apparent on-rate for the tonic block increases with the prepulse voltage in the range -60 to -45 mV, where significant gating current but no ionic current occurs. When plotted together against voltage, the on-rates of tonic block (-100 to -45 mV) and of use-dependent block (-40 to 40 mV) fall on a single sigmoid that parallels the voltage dependence of the gating charge. The on-rate of tonic block by Gd(3+) decreases with concentration of Ba(2+), indicating that the apparent affinity of the site to permeant ions is about 1 mM in closed channels. Therefore, we propose that at submicromolar concentrations, Gd(3+) binds at the entry to the selectivity locus and that the affinity of the site for permeant ions decreases during preopening transitions of the channel.     

Interactions of monovalent cations with sodium channels in squid axon. II. Modification of pharmacological inactivation gating

The time-, frequency-, and voltage-dependent blocking actions of several cationic drug molecules on open Na channels were investigated in voltage-clamped, internally perfused squid giant axons. The relative potencies and time courses of block by the agents (pancuronium [PC], octylguanidinium [C8G], QX-314, and 9-aminoacridine [9-AA]) were compared in different intracellular ionic solutions; specifically, the influences of internal Cs, tetramethylammonium (TMA), and Na ions on block were examined. TMA+ was found to inhibit the steady state block of open Na channels by all of the compounds. The time-dependent, inactivation-like decay of Na currents in pronase-treated axons perfused with either PC, 9-AA, or C8G was retarded by internal TMA+. The apparent dissociation constants (at zero voltage) for interaction between PC and 9-AA with their binding sites were increased when TMA+ was substituted for Cs+ in the internal solution. The steepness of the voltage dependence of 9-AA or PC block found with internal Cs+ solutions was greatly reduced by TMA+, resulting in estimates for the fractional electrical distance of the 9-AA binding site of 0.56 and 0.22 in Cs+ and TMA+, respectively. This change may reflect a shift from predominantly 9-AA block in the presence of Cs+ to predominantly TMA+ block. The depth, but not the rate, of frequency-dependent block by QX-314 and 9-AA is reduced by internal TMA+. In addition, recovery from frequency-dependent block is not altered. Elevation of internal Na produces effects on 9-AA block qualitatively similar to those seen with TMA+. The results are consistent with a scheme in which the open channel blocking drugs, TMA (and Na) ions, and the inactivation gate all compete for a site or for access to a site in the channel from the intracellular surface. In addition, TMA ions decrease the apparent blocking rates of other drugs in a manner analogous to their inhibition of the inactivation process. Multiple occupancy of Na channels and mutual exclusion of drug molecules may play a role in the complex gating behaviors seen under these conditions.     

Kinetics of 9-aminoacridine block of single Na channels

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels.

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.