Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Sodium-dependent activation of intestinal brush-border sucrase: Correlation with activation by deprotonation from pH 5 to 7

The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK₁ of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK₂ of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V_m and K_m. The V_m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK_1y, about 5.6) was little affected by sodium. On the contrary, the K_m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK_1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541).     

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.     

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na⁺ in the external solution by K⁺ or NH ₄ ⁺ the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — P_Na: \(P_{NH_4 }\) :P_K=1:0.47:0.19. The relative permeability for H⁺ was determined by measuring currents after replacement of Na⁺ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio p_H/p_Na for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H⁺ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.     

Functional Expression of Drosophila para Sodium Channels : Modulation by the Membrane Protein TipE and Toxin Pharmacology

The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (K _d ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.     

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T

We have investigated the effects of a mild oxidant, chloramine-T(CT), on the sodium and potassium currents of squid axons under voltage-clamp conditions. Sodium channel inactivation of squid giant axons can be completely removed by CT at neutral pH. Internal and external CT treatment are both effective. CT apparently removes inactivation in an irreversible, all-or-none manner. The activation process of sodium channels is little affected, as judged from the voltage dependence of peak sodium currents, the rising phase of sodium currents, and the time course of tail currents following the repolarization. The removal of inactivation by CT is pH-dependent; higher pH decreases the removal rate, whereas lower pH increases it. Internal metabisulfite, a strong reductant, does not protect inactivation from the action of external CT, nor does external metabisulfite protect from internal CT application. CT slightly depresses the peak potassium currents at comparable concentrations but has no apparent effects on their kinetics. Our results suggest that the neutral form of CT modifies an embedded methionine residue that is involved in sodium channel inactivation.     

Interaction of the Depolarization-Activated K Channel of Samanea saman with Inorganic Ions: A Patch-Clamp Study

A depolarization-activated K⁺ channel capable of carrying the large K⁺ currents that flow from shrinking cells during movements of Samanea saman leaflets has been described in the plasmalemma of Samanea motor cell protoplasts (N Moran et al [1988] Plant Physiol 88:643-648). We now characterize this channel in greater detail. It is selective for K⁺ over other monovalent ions, with the following order of relative permeability: K⁺ > Rb⁺ > Na⁺ Cs⁺ Li⁺. It is blocked by Cs⁺ and by Ba²⁺ in a voltage dependent manner, exhibiting a `long-pore' behavior, similarly to various types of K⁺ channels in animal systems. Cadmium, known for its blockage of Ca²⁺ channels in animal systems, and Gd³⁺, closely related to La³⁺, which also blocks Ca²⁺ channels in animal cells, both block K⁺ currents in Samanea in a voltage-independent manner, and without interfering with the kinetics of the currents. The suggested mechanism of block is either (a) by a direct interaction with the K⁺ channel, but external to its lumen, or, alternatively, (b) by blocking putative Ca²⁺ channels, and preventing the influx of Ca²⁺, on which the activation of the K⁺ channels may be dependent.     

Block of sodium conductance and gating current in squid giant axons poisoned with quaternary strychnine.

Quaternary strychnine blocks sodium channels from the axoplasmic side, probably by insertion into the inner channel mouth. Block is strongly voltage dependent, being more pronounced in depolarized than in resting axons. Using potential steps as a means to modulate the level of block, we investigate strychnine effects on sodium and gating currents at +50 and -50 mV. We analyze our data in terms of the simplest possible model, wherein only an open channel may receive and retain a strychnine molecule. Our main findings are (a) block by strychnine and inactivation resemble each other and (b) block of sodium and gating currents by strychnine happen with closely similar time-courses. Our data support the hypothesis of Armstrong and Bezanilla (1977) wherein an endogenous blocking particle causes inactivation by inserting itself into the inner mouth of the sodium channel. Quaternary strychnine may act as an artificial substitute for the hypothetical endogenous blocking particle. Further, we suggest that at least 90% of the rapid asymmetrical displacement current in squid axons is sodium channel gating current, inasmuch as quaternary strychnine can block 90% of the displacement current simultaneously with sodium current.     

Sensitivity of the sodium and potassium channels of Myxicola giant axons to changes in external pH

Myxicola giant axons were studied using standard voltage-clamp techniques in solutions whose pH values ranged from 3.9 to 10.2. Buffer concentrations of 50 mM or greater were necessary to demonstrate the full effect of pH. In acidic solutions the axon underwent a variable depolarization, and both the sodium and potassium conductances were reversibly depressed with approximate pKa's of 4.8 and 4.4, respectively. The voltage dependence of GNa was only slightly altered by acidic conditions, whereas there occurred large shifts in GK along the voltage axis consistent with a substantial decrease in net negative surface charge in the vicinity of the K+ channels. The sodium and potassium activation rate constants were decreased by acidic conditions, but the results could not be described as a simple translation along the voltage axis.     

Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P _o). Although P _o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca²⁺ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb⁺ is substituted for K⁺ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P _o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

Closed-channel block of BK potassium channels by bbTBA requires partial activation

Blockade of large-conductance Ca²⁺-activated K⁺ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca²⁺-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca²⁺ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca²⁺; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.     

Inhibition of sodium currents by local anesthetics in chloramine-T- treated squid axons. The role of channel activation

In order to test the requirement of Na channel inactivation for the action of local anesthetics, we investigated the inhibitory effects of quaternary and tertiary amine anesthetics on normally inactivating and noninactivating Na currents in squid axons under voltage clamp. Either the enzymatic mixture pronase, or chloramine-T (CT), a noncleaving, oxidizing reagent, was used to abolish Na channel inactivation. We found that both the local anesthetics QX-314 and etidocaine, when perfused internally at 1 mM, elicited a "tonic" (resting) block of Na currents, a "time-dependent" block that increased during single depolarizations, and a "use-dependent" (phasic) block that accumulated as a result of repetitive depolarizations. All three effects occurred in both control and CT-treated axons. As in previous reports, little time-dependent or phasic block by QX-314 appeared in pronase-treated axons, although tonic block remained. Time-dependent block was greatest and fastest at large depolarizations (Em greater than +60 mV) for both the control and CT-treated axons. The recovery kinetics from phasic block were the same in control and CT-modified axons. The voltage dependence of the steady state phasic block in CT-treated axons differed from that in the controls; an 8-10% reduction of the maximum phasic block and a steepening and shift of the voltage dependence in the hyperpolarizing direction resulted from CT treatment. The results show that these anesthetics can bind rapidly to open Na channels in a voltage-dependent manner, with no requirement for fast inactivation. We propose that the rapid phasic blocking reactions in nerve are consequences primarily of channel activation, mediated by binding of anesthetics to open channels, and that the voltage dependence of phasic block arises directly from that of channel activation.     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.     

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K⁺ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K⁺ channel ligands can expand in the subunit interface whose residues are less conserved between K⁺ channels and hence may be targets for design of highly desirable subtype-specific K⁺ channel drugs.     

Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide

The group-specific protein reagents, N-bromacetamide (NBA) and N- bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur- containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K_Ca2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K_Ca2.2 and K_Ca2.3 with the same high affinity (K_D ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC₅₀ values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K_Ca2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

Dependence of ion flow through the hemocyanin channel on a fixed charge at the pore mouth: effects of H + and Ca2+ ions

Incorporation of Megatura crenulata hemocyanin into planar phospholipid bilayers results in the formation of ionic channels whose conductance can be directly measured. We have studied the effects of the pH on the electrical properties of these channels in the presence both of a K₂SO₄ solution, at high and low concentration, and of a KCl one. We have found that the conductance of the channel depends on the proton concentration following a positive titration curve, i.e., increasing sigmoidally with the pH at all the concentrations used; at any given pH, it additionally increases sublinearly with the concentration of the salt. The sublinear conductance-concentration dependence can be reverted to an almost linear one by the addition of suitable amounts of an indifferent cation such a tetramethylammonium to keep the ionic strength constant. The current-voltage curve of the channel, which is strongly voltage-dependent, is shifted along the voltage axis towards negative values by an increase in the proton concentration. Calcium ions have similar effects. The selectivity of the channel for cations over anions is strongly pH-dependent in the case of a KCl solution, being lost at pH 4.5, but is almost invariant in a K₂SO₄ solution. All experimental results are interpreted assuming the existence of a mechanism of voltage gating of the channel and of discrete negative charge fixed near its mouth. This charge can be neutralized by specific binding either of H⁺ or of Ca²⁺ ions. The dissociation constants from the channel found for these two ions are consistent with those given in the literature for the hemocyanin protein and indicate that carboxyl groups and/or histidines are involved in forming the negative charge of the pore.