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H A Fozzard

Publications and source records attributed to H A Fozzard.

At least 19 recordsLinked to original sources
PubMed2006

Isoform-dependent interaction of voltage-gated sodium channels with protons.

Protons are potent physiological modifiers of voltage-gated Na(+) channels, shifting the voltage range of channel gating and reducing current magnitude (pK(a) approximately 6). We recently showed that proton block of the skeletal muscle isoform (Na(V)1.4) resulted from protonation of the four superficial carboxylates in the outer vestibule of the channel. We concluded that the large local negative electrostatic field shifted the outer vestibule carboxylate pK(a) into the physiological range. However, block was not complete; the best-fit titration curves yielded an acid pH asymptote of 10-15%, suggesting that the selectivity filter carboxylates may not be protonated. Using HEK 293 cells stably expressing different isoforms, each with varying channel density, we demonstrate that a pH-independent current is found in Na(V)1.4, but not in the cardiac isoform (Na(V)1.5). Mutational studies showed that absence of the pH-independent current in Na(V)1.5 could be ascribed to the cysteine in domain I, just above the selectivity filter aspartate (Cys373). We suggest that this cysteine can be protonated in acid solution to produce a positive charge that blocks the pore. Competition between protons and Na(+) did not exist for Na(+) concentrations between 1 and 140 mm. The residual current in acid solution, when the cysteine is absent, confirms that over the range of pH values that can be achieved physiologically, the selectivity filter carboxylates are not protonated. The pH-independent current helps to protect activation of skeletal muscle during the acidosis that occurs during exercise.

Acidosis↗
PubMed2005

Mechanism of local anesthetic drug action on voltage-gated sodium channels.

Local anesthetic drugs interfere with excitation and conduction by action potentials in the nervous system and in the heart by blockade of the voltage-gated Na channel. Drug affinity varies with gating state of the channel. The drugs show low affinity at slow excitation rates, but high affinity when the channels are opened and inactivated during action potentials at high frequency, as they are during pain or during a cardiac arrhythmia. The drugs are thought to access their binding site in the inner pore by passage through the membrane and entry through the inner pore vestibule. There have been three major developments in the last decade that greatly increase our understanding of their mechanism of action. Firstly, amino acid residues critical to drug binding have been located by mutagenesis, and it is possible to develop a molecular model of the drug binding site. Secondly, a path for drug access directly from the outside has been characterized in the cardiac isoform of the channel. Thirdly, the hypothesis that high affinity binding stabilizes the fast inactivated conformation of the channel has been challenged. Rather, the drug may stabilize a slow inactivated state and immobilize the voltage sensor in domain III in its activated outward position. The combination of mutational study of the cloned Na channels and patch clamp offers the opportunity to understand the detailed molecular mechanism of drug action and to resolve drug structure-function.

Anesthetics, Local↗
PubMed2002

Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block.

Voltage-gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure-function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Na(v)1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two-micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half-activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pK(a) (-log of the dissociation constant) in the acid direction. Effects of neutralization on pK(a) were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pK(a) values of carboxylates of Glu-403, Glu-758, and Asp-1532 are shifted to values similar to the experimentally measured pK(a). Calculations also predict the experimentally observed changes in pK(a) that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.

Animals↗
PubMed2001

Cardiac-specific external paths for lidocaine, defined by isoform-specific residues, accelerate recovery from use-dependent block.

Local anesthetic antiarrhythmic drugs block voltage-gated Na(+) channels from the cytoplasmic side. In addition, cardiac Na(+) channels can be also blocked by the membrane-impermeant local anesthetic QX via external paths not present in skeletal muscle or brain channels. Introduction of cardiac isoform-specific residues into wild-type skeletal muscle or brain channels creates access paths for external QX block. These paths should affect the characteristics of use-dependent block by influencing drug on- and off-rates. We investigated the effects of these external paths on drug kinetics of lidocaine, a lipophilic drug of clinical relevance, by studying use-dependent block using a two-electrode voltage clamp in Xenopus oocytes. Recovery from use-dependent block was slowed when cardiac isoform-specific residues important for external QX access were mutated to skeletal muscle or brain isoform-specific residues. As the fraction of charged lidocaine was decreased by raising external pH, differences in recovery kinetics diminished, indicating that these mutations mostly influenced block by charged lidocaine molecules. Data were fit into a model in which bound drug distributes into charged and neutral forms based on its pK(a) and external pH with separate dissociation paths and recovery-time constants. These isoform-specific mutations altered the recovery-time constants for the charged molecules with smaller effects on those for the neutral molecules. We conclude that the external egress paths created by isoform-specific residues influence the drug kinetics of lidocaine, and these residues define cardiac-specific external paths for local anesthetic drugs.

Animals↗
PubMed2001

Modeling of the outer vestibule and selectivity filter of the L-type Ca2+ channel.

Using the KcsA bacterial K+ channel crystal structure [Doyle, D. A., et al. (1998) Science 280, 69-74] and the model of the outer vestibule of the Na+ channel [Lipkind, G. M., and Fozzard, H. A. (2000) Biochemistry 39, 8161-8170] as structural templates, we propose a structural model of the outer vestibule and selectivity filter of the pore of the Ca2+ channel (alpha1C or Ca(v)1.2). The Ca2+ channel P loops were modeled by alpha-helix-turn-beta-strand motifs, with the glutamate residues of the EEEE motif located in the turns. P loops were docked in the extracellular part of the inverted teepee structure formed by S5 and S6 alpha-helices with backbone coordinates from the M1 and M2 helices of the KcsA crystal structure. This construction results in a conical outer vestibule that tapers to the selectivity filter at the bottom. The modeled selectivity ring forms a wide open pore ( approximately 6 A) in the absence of Ca2+. When Ca2+ is present ( approximately 1 microM), all four glutamate side chains move to the center and form a cage around the dehydrated Ca2+ ion, blocking the pore. In the millimolar concentration range, Ca2+ also interacts with two low-affinity sites located externally and internally, which were modeled by the same carboxylate groups of the selectivity filter. Calculation of the resulting electrostatic potentials show that the single Ca2+ ion is located in an electrostatic trap. Only when three Ca2+ ions are bound simultaneously in the high- and low-affinity sites of the selectivity filter is Ca2+ able to overcome electrostatic attraction, permitting Ca2+ flux.

Amino Acid Motifs↗
PubMed2001

The selectivity filter of the voltage-gated sodium channel is involved in channel activation.

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultra-slow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of approximately 100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant mu-conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D.

3' Untranslated Regions↗
PubMed2001

Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis.

The voltage-gated Na+ channel alpha-subunit consists of four homologous domains arranged circumferentially to form the pore. Several neurotoxins, including saxitoxin (STX), block the pore by binding to the outer vestibule of this permeation pathway, which is composed of four pore-forming loops (P-loops), one from each domain. Neosaxitoxin (neoSTX) is a variant of STX that differs only by having an additional hydroxyl group at the N1 position of the 1,2,3 guanidinium (N1-OH). We used this structural variant in mutant cycle experiments to determine interactions of the N1-OH and its guanidinium with the outer vestibule. NeoSTX had a higher affinity for the adult rat skeletal muscle Na+ channel (muI or Scn4a) than for STX (DeltaG approximately = 1.3 kcal/mol). Mutant cycle analysis identified groups that potentially interacted with each other. The N1 toxin site interacted most strongly with muI Asp-400 and Tyr-401. The interaction between the N1-OH of neoSTX and Tyr-401 was attractive (DeltaDeltaG = -1.3 +/- 0.1 kcal/mol), probably with formation of a hydrogen bond. A second possible attractive interaction to Asp-1532 was identified. There was repulsion between Asp-400 and the N1-OH (DeltaDeltaG = 1.4 +/- 0.1 kcal/mol), and kinetic analysis further suggested that the N1-OH was interacting negatively with Asp-400 at the transition state. Changes in pH altered the affinity of neoSTX, as would be expected if the N1-OH site were partially deprotonated. These interactions offer an explanation for most of the difference in blocking efficacy between neoSTX and STX and for the sensitivity of neoSTX to pH. Kinetic analysis suggested significant differences in coupling energies between the transition and the equilibrium, bound states. This is the first report to identify points of interaction between a channel and a non-peptide toxin. This interaction pattern was consistent with previous proposals describing the interactions of STX with the outer vestibule (Lipkind, G. M., and H. A. Fozzard. 1994. Biophys. J. 66:1-13; Penzotti, J. L., G. Lipkind, H. A. Fozzard, and S. C. Dudley, Jr. 1998. Biophys. J. 75:2647-2657).

Animals↗
PubMed2001

Structural and gating changes of the sodium channel induced by mutation of a residue in the upper third of IVS6, creating an external access path for local anesthetics.

Membrane-impermeant quaternary amine local anesthetics QX314 and QX222 can access their binding site on the cytoplasmic side of the selectivity filter from the outside in native cardiac Na(+) channels. Mutation of domain IV S6 Ile-1760 of rat brain IIA Na(+) channel or the equivalent (Ile-1575) in the adult rat skeletal muscle isoform (mu 1) creates an artificial access path for QX. We examined the characteristics of mutation of mu 1-I1575 and the resulting QX path. In addition to allowing external QX222 access, I1575A accelerated decay of Na(+) current and shifted steady-state availability by -27 mV. I1575A had negligible effects on inorganic or organic cation selectivity and block by tetrodotoxin (TTX), saxitoxin (STX), or mu-conotoxin (mu-CTX). It exposed a site within the protein that binds membrane-permeant methanethiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethiosulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsulfonate (MTSES). MTSEA binding abolished the QX path created by this mutation, without effects on toxin binding. The mu-CTX derivative R13N, which partially occluded the pore, had no effect on QX access. I1575A exposed two Cys residues because a disulfide bond was formed under oxidative conditions, but the exposed Cys residues are not those in domain IV S6, adjacent to Ile-1575. The Cys mutant I1575C was insensitive to external Cd(2+) and MTS compounds (MTSEA, MTSET, MTSES), and substitution of Ile with a negatively charged residue (I1575E) did not affect toxin binding. Ile-1575 seems to be buried in the protein, and its mutation disrupts the protein structure to create the QX path without disturbing the outer vestibule and its selectivity function.

Amino Acid Substitution↗
PubMed2000

KcsA crystal structure as framework for a molecular model of the Na(+) channel pore.

The crystal structure of the pore-forming part of the KcsA bacterial K(+)-selective channel suggests a possible motif for related voltage-gated channels. We examined the hypothesis that the spacial orientation of the KcsA M1 and M2 alpha-helices also predicts the backbone location of S5 and S6 helices of the voltage-gated Na(+) channel. That channel's P region structure is expected to be different because selectivity is determined by side-chain interactions rather than by main-chain carbonyls, and its outer vestibule accommodates relatively large toxin molecules, tetrodotoxin (TTX) and saxitoxin (STX), which interact with selectivity ring residues. The Na(+) channel P loop was well-modeled by the alpha-helix-turn-beta-strand motif, which preserves the relationships for toxin interaction with the Na(+) channel found experimentally. This outer vestibule was docked into the extracellular part of the inverted teepee structure formed by the S5 and S6 helices that were spacially located by coordinates of the KcsA M1 and M2 helix main chains [Doyle et al. (1998) Science 280, 69-74], but populated with side chains of the respective S5 and S6 structures. van der Waals contacts were optimized with minimal adjustment of the S5, S6, and P loop structures, forming a densely packed pore structure. Nonregular external S5-P and P-S6 segments were not modeled here, except the P-S6 segment of domain II. The resulting selectivity region structure is consistent with Na(+) channel permeation properties, offering suggestions for the molecular processes involved in selectivity. The ability to construct a Na(+) channel pore model consistent with most of the available biophysical and mutational information suggests that the KcsA structural framework may be conserved in voltage-gated channels.

Amino Acid Sequence↗
PubMed2000

A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel.

Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na(+) channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle (micro1) and rat heart (rH1) Na(+) channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the micro1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. mu1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in mu1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant (mu1-Y401C/F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of mu1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in mu1-Y401C was more than that in mu1-C1572T, and the double mutant (mu1-Y401C/C1572T) accelerated internal QX recovery more than mu1-Y401C or mu1-C1572T alone. We conclude that the isoform-specific residue (Tyr/Phe/Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.

Anesthetics, Local↗
PubMed2000

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains.

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

Amino Acids↗
PubMed1999

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule.

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.

Animals↗
PubMed1998

Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis.

High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the critical Arg-13 and amino acid residues known to be in the channel's outer vestibule. Arg-13 interacts specifically with domain II Glu-758 with energy of about -3.0 kcal/mol, including both electrostatic and nonelectrostatic components, and with Glu-403 with energy of about -2.0 kcal/mol. Interactions with the other charged residues in the outer vestibule were shown to be almost entirely electrostatic, because these interactions were maintained when Arg-13 was replaced by lysine. These results place the bound Arg-13 at the channel mouth adjacent to the P (pore) loops of domains I and II. Distance estimates based on interaction energies suggest that the charged vestibule residues are in relative positions similar to those of the Lipkind-Fozzard vestibule model [Lipkind, G. M., and Fozzard, H. A. (1994) Biophys. J. 66, 1-13]. Kinetic analysis suggests that Arg-13 interactions are partially formed in the ligand-channel transition state.

Animals↗
PubMed1998

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.

Adult↗
PubMed1997

Sodium channel selectivity filter regulates antiarrhythmic drug binding.

Local anesthetic antiarrhythmic drugs block Na+ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel's selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na+ channel (micro1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na+ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.

Anesthetics, Local↗
PubMed1997

A model of scorpion toxin binding to voltage-gated K+ channels.

Mutational studies have identified part of the S5-S6 loop of voltage-dependent K+ channels (P region) responsible for tetraethylammonium (TEA) block and permeation properties. Several scorpion peptide toxins-charybdotoxin (ChTX), kaliotoxin (KITX), and agitoxin (AgTX)-also block the channel with high affinity and specificity. Here, we examine the interaction predicted when the toxins are docked onto the molecular model of the K+ channel pore that we recently proposed. Docking with the model of the Kv1.3 channel started by location of Lys-27 side chain into the central axis of the pore, followed by energy minimization. In the optimal arrangement, Arg-24 of KITX or AgTX forms a hydrogen bond with the Asp-386 carboxyl of one subunit, and Asn-30 is in immediate contact with Asp-386 of the opposing subunit in the tetramer. Toxin residues in proximity to the side chain of Lys-27 (Phe-25, Thr-36, Met-29, and Ser-11 in KITX) interact with the four C-end His-404s. For ChTX the interaction with Asp-386 is reduced, but this is compensated by additional nonbonded interactions formed by Tyr-36 and Arg-34. Comparison of calculated energy of interaction of these specific toxin-channel residues with experimental studies reveals good agreement. The similar total calculated energy of interaction is consistent with the similar IC50 for Kv1.3 block by KITX and AgTX. Steric contacts of residues in position 380 of the S5-P linker with residues on the upper part of toxins permit reconstruction of the K+ channel outer vestibule walls, which are about 30 A apart and about 9 A high. Molecular modeling shows complementarity of the pore model to toxin spacial structures, and supports the proposal that the N-terminal borders of the P regions surround residues of their C-terminal halves.

Amino Acid Sequence↗