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J A Drewe

Publications and source records attributed to J A Drewe.

At least 19 recordsLinked to original sources
PubMed2001

Pentylenetetrazole-induced inhibition of recombinant gamma-aminobutyric acid type A (GABA(A)) receptors: mechanism and site of action.

Pentylenetetrazole (PTZ) is a central nervous system convulsant that is thought, based on binding studies, to act at the picrotoxin (PTX) site of the gamma-aminobutyric acid type A (GABA(A)) receptor. In the present study, we have investigated the mechanism and site of action of PTZ in recombinant GABA(A) receptors. In rat alpha 1 beta 2 gamma 2 receptors, PTZ inhibited GABA-activated Cl(-) current in a concentration-dependent, voltage-independent manner, with an IC(50) of 0.62 +/- 0.13 mM. The mechanism of inhibition appeared competitive with respect to GABA in both rat and human alpha 1 beta 2 gamma 2 receptors. Varying subunit configuration (change or lack of alpha subunit isoform or lack of gamma 2 subunit) had modest effects on PTZ-induced inhibition, as evidenced by comparable IC(50) values (0.6-2.2 mM) in all receptor configurations tested. This contrasts with PTX and other PTX-site ligands, which have greater affinity in receptors lacking an alpha subunit. Using a one-site model for PTZ interaction with alpha 1 beta 2 gamma 2 receptors, the association rate (k(+1)) was found to be 1.14 x 10(3) M(-1) s(-1) and the dissociation rate (k(-1)) was 0.476 s(-1), producing a functional k(d) of 0.418 mM. PTZ could only gain access to its binding site extracellularly. Single-channel recordings demonstrated that PTZ decreased open probability by increasing the duration of closed states but had no effect on single-channel conductance or open state duration. alpha-Isopropyl-alpha-methyl-gamma-butyrolactone, a compound known to antagonize effects of PTX, also diminished the effects of PTZ. Taken together, our results indicate that pentylenetetrazole and picrotoxin interact with overlapping but distinct domains of the GABA(A) receptor.

Algorithms↗
PubMed2000

Influence of subunit configuration on the interaction of picrotoxin-site ligands with recombinant GABA(A) receptors.

We have assessed the interaction of picrotoxin and a putative picrotoxin-site ligand [4-dimethyl-3-t-butylcarboxyl-4,5-dihydro (1, 5-a) quinoxaline] (U-93631) with varying configurations of recombinant GABA(A) receptors, using the whole-cell patch clamp technique. In alpha2beta2gamma2 GABA(A) receptors, coapplication of picrotoxin with GABA had minimal effects on initial GABA-activated Cl(-) current amplitude, and subsequently enhanced decay of GABA-activated Cl(-) currents. The half-maximal inhibitory concentration (IC(50)) for picrotoxin in alpha2beta2gamma2 receptors was 10.3+/-1.6 microM. The alpha subunit isoform did not affect picrotoxin-induced inhibition, as IC(50) values for alpha3beta2gamma2 (5.1+/-0.7 microM) and alpha6beta2gamma2 receptors (7.2+/-0.4 microM) were comparable to those obtained in alpha2beta2gamma2 receptors. Interestingly, in receptors lacking an alpha subunit (beta2gamma2 configuration), picrotoxin had a markedly lower IC(50) (0.5+/-0.05 microM) compared to alpha-containing receptors. The inhibitory profile was generally similar for the presumed picrotoxin-site ligand U-93631, i.e., IC(50) values were comparable in all alphabetagamma-containing receptors, but the IC(50) in beta2gamma2 receptors was greater than 10-fold lower. In addition, a modest but significant initial stimulation of GABA-activated current by U-93631 was observed in alpha2beta2gamma2 and beta2gamma2 receptors. A mutation in the second transmembrane domain, shown previously to abolish picrotoxin sensitivity, also greatly attenuated sensitivity to U-93631. Moreover, incubation of receptors with excess U-93631 hindered picrotoxin's ability to gain access to its binding site; both results indicate that U-93631 interacts at the picrotoxin site of the receptor. Our results indicate the presence of an alpha subunit hinders the ability of picrotoxin to block the GABA(A) receptor, and thus provides additional insight into the site of action of picrotoxin. In addition, we have shown that domains important for the actions of picrotoxin also affect U-93631. Thus, this compound should prove to be a useful ligand for analysis of the convulsant site of this receptor.

Amino Acid Substitution↗
PubMed1998

Structure-activity relationships for a series of bis(phenylalkyl)amines: potent subtype-selective inhibitors of N-methyl-D-aspartate receptors.

A series of bis(phenylalkyl)amines, structural analogues of ifenprodil and nylidrin, were synthesized and tested for antagonism of N-methyl-D-aspartate (NMDA) receptors. Potency and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing three binary combinations of cloned rat NMDA receptor subunits: NR1A expressed in combination with either NR2A, NR2B, or NR2C. The bis(phenylalkyl)amines were selective antagonists of NR1A/2B receptors. Assayed under steady-state conditions, the most potent of these, N-[2-(4-hydroxyphenyl)ethyl]-5-phenylpentylamine hydrochloride (20), has an IC50 value of 8 nM and >1000-fold selectivity with respect to NR1A/2A and NR1A/2C receptors. The structure-activity relationship of the bis(phenylalkyl)amine series indicates that the piperidine ring and alkyl chain substitutions common to NR2B-selective antagonists such as ifenprodil, CP 101,606, and Ro 25-6981 are not necessary to generate potent and selective ligands. The primary determinants of potency are the phenolic OH group, acting as a hydrogen bond donor, the distance between the two rings, and an electrostatic interaction between the receptor and the basic nitrogen atom. This study provides a framework for designing structurally novel NR2B-selective antagonists which may be useful for treatment of a variety of neurological disorders.

Animals↗
PubMed1998

Substituted 3beta-phenylethynyl derivatives of 3alpha-hydroxy-5alpha-pregnan-20-one: remarkably potent neuroactive steroid modulators of gamma-aminobutyric acidA receptors.

Neuroactive steroids are positive allosteric modulators of gamma-aminobutyric acidA (GABAA) receptor complexes. Synthetic modification generally does not increase neuroactive steroid potency beyond that of the naturally occurring progesterone metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P). Recently, it has been shown that introduction of appropriately para-substituted phenylethynyl groups at the 3beta-position of 5beta steroids increases receptor potency. The present report presents the synthesis and pharmacological profile of an analogous series of 5alpha steroids. The most striking feature of this series is the further enhancement of in vitro and in vivo potency obtained. In particular, 3beta-(p-acetylphenylethynyl)-3alpha-hydroxy-5alpha-pr egnan-20-one (Co 152791) was 11-, 16- and 49-fold more potent than 3alpha, 5alpha-P in modulating the binding of [35S]TBPS, [3H]flunitrazepam and [3H]muscimol, respectively, in rat brain membranes (Co 152791 IC50 or EC50 = 2-7.5 nM). Similarly, Co 152791 was 3- to 20-fold more potent than 3alpha,5alpha-P as an inhibitor of [35S]TBPS binding in human recombinant receptor combinations containing alpha1, alpha2, alpha3 or alpha5 and beta2gamma2L subunits (Co 152791 IC50 1.4-5.7 nM). Co 152791 displayed low efficacy and 3alpha,5alpha-P had low potency at alpha4/6beta3gamma2L GABAA receptor complexes. Interestingly, Co 152791 demonstrated remarkable potency as a potentiator of GABA-evoked currents in Xenopus oocytes expressing alpha1beta2gamma2L receptors (EC50 0.87 nM), being 184-fold more potent than 3alpha,5alpha-P. High in vitro potency was also reflected in enhanced in vivo activity in that Co 152791 exhibited exceptional anticonvulsant potency, protecting mice from pentylenetetrazol-induced seizures at a approximately 5-fold lower dose than 3alpha,5alpha-P after i.p. administration (Co 152791 ED50 0.6 mg/kg). Moreover, Co 152791 was orally active (ED50 1.1 mg/kg) and exhibited a therapeutic index of 7 relative to rotorod impairment. The remarkable potency of Co 152791 as a positive allosteric modulator of GABAA receptors may be explained by its interaction with an auxiliary binding pocket in the neuroactive steroid binding site. In addition, modification at the 3beta-position probably hinders metabolism of the 3alpha-hydroxy group contributing to the exceptional anticonvulsant potency of this compound relative to other neuroactive steroids.

Animals↗
PubMed1996

3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor.

Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors.

Allosteric Regulation↗
PubMed1996

Pharmacology of the human gamma-aminobutyric acidA receptor alpha 4 subunit expressed in Xenopus laevis oocytes.

The human gamma-aminobutyric acidA (GABAA) receptor alpha 4 subunit was recently cloned and characterized pharmacologically using radioligand binding techniques. These studies suggested that alpha 4 subunits confer a novel diazepam-insensitive binding site. To further investigate the pharmacology of the alpha 4 subunit, we expressed human alpha 4 beta 2 gamma 2L subunit combinations in oocytes and compared the expression and pharmacology of these receptors with alpha 1 beta 2 gamma 2L, beta 2 gamma 2L, and other possible binary subunit combinations. Apparent GABA affinity was 2-3-fold higher for alpha 4 beta 2 gamma 2L than for alpha 1 beta 2 gamma 2L receptors. Functional modulation of receptors by benzodiazepine-site ligands and other classes of allosteric modulator were assayed over a broad concentration range (0.01-100 microM) on currents that were 10% of the maximum GABA response. Diazepam (0.01-1 microM) did not modulate GABA responses at alpha 4 beta 2 gamma 2L receptors, whereas it increased alpha 1 beta 2 gamma 2L responses by approximately 110%. Bretazenil (0.01-1 microM), a benzodiazepine partial agonist, induced higher efficacy modulation of alpha 4 beta 2 gamma 2L receptors (approximately 83%) than of alpha 1 beta 2 gamma 2L (approximately 25%). The benzodiazepine antagonist flumazenil (0.1-10 microM) unexpectedly potentiated alpha 4 beta 2 gamma 2L responses up to approximately 41%, and the benzodiazepine partial inverse agonist Ro15-4513 (1 microM) potentiated alpha 4 beta 2 gamma 2L responses by approximately 63%. Two other benzodiazepine-site ligands, CGS-9895 and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, had qualitatively similar effects at alpha 1 beta 2 gamma 2L and alpha 4 beta 2 gamma 2L. Modulators such as pentobarbital, 3 alpha-hydroxy-5 alpha-pregnan-20-one, mefenamic acid, and loreclezole also induced similar potentiation at both subtypes of receptor. The pharmacology conferred by the alpha 4 subunit was similar to that conferred by the alpha 6 subunit, to which it shows highest levels of homology, but the two subunits differ in sensitivity to the beta-carboline methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. Properties of the alpha 4-containing receptors are consistent with diazepam-insensitive binding sites found in cerebral cortex and other forebrain structures. Characterization of these receptors should further our understanding of mechanisms underlying the behavioral effects of GABA modulators and help in the design of drugs with improved, or novel, therapeutic profiles.

Animals↗
PubMed1995

Cloning and characterization of the human GABAA receptor alpha 4 subunit: identification of a unique diazepam-insensitive binding site.

Benzodiazepines modulate gamma-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABAA receptor-chloride channel complex. The heterogeneity of diazepam-sensitive benzodiazepine binding sites (type I and type II) has been identified by pharmacological approaches both with native receptors and recombinant receptors coexpressing alpha, beta and gamma subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites are found, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of alpha 6 with beta 2 and gamma 2L subunits creates a pharmacologically similar benzodiazepine receptor to the diazepam-insensitive site observed in cerebellum, however, there is no evidence regarding the possible subunit combination forming the DI site in cerebral tissues. Here we report the cloning of the human alpha 4 cDNA and its pharmacology by coexpression of this alpha 4 subunit with beta 2 and gamma 2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenill, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-beta-carboline-3-carboxylate) and Ro15-4513 as determined by [3H]Ro15-4513 binding. However, it is insensitive to the benzodiazepine type I selective compounds CL218.872 (3-methyl-6-[3-(trifluoromethyl)[phenyl]-1,2,4-triazolo[4.3-b]pyridazine ) and zolpidem as well as the benzodiazepine full agonists diazepam, halazolam and midazolam. In addition, the benzodiazepine receptor ligands DMCM, beta-CCE (beta-carboline-3-carboxylate ethyl ester), Beta-CCM (beta-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for alpha 4 beta 2 gamma 2L. The pharmacology of the alpha 4 beta 2 gamma 2L receptor complex appears to resemble those of the diazepam-insensitive site found in the cerebral cortex. Our study thus suggests that this subpopulation of diazepam-insensitive GABAA receptors may be composed of alpha 4 beta 2 gamma 2L subunits.

Amino Acid Sequence↗
PubMed1995

Stable high expression of human gamma-aminobutyric acidA receptors composed of alpha and beta subunits.

Multiple classes of pharmacological agents including benzodiazepines, cage convulsants like t-butylbicyclophosphorothionate (TBPS), barbiturates and neuroactive steroids allosterically modulate the gamma-aminobutyric acidA receptor-chloride ionophore complex (GRC). The function of benzodiazepines requires a GRC comprised of alpha, beta and gamma subunits, while TBPS, barbiturates and neuroactive steroids will allosterically modulate GRCs comprised of only alpha and beta subunits. Binary alpha beta complexes are still hypothesized to be expressed in the mammalian brain particularly during development and could contribute to the pharmacological action of neuroactive steroids and barbiturates. In order to examine binary alpha beta complexes we report here the establishment of stable cell lines that express high levels of human GABAA receptors comprised of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 subunit combinations. The apparent potencies for allosteric modulation of [35S]TBPS for most naturally occurring neuroactive steroids for the binary subunit combinations was similar to that of the gamma-containing subunit combinations. Also discussed is the usefulness of these cell lines for the biophysical analysis of the GABAA receptor stoichiometry.

Animals↗
PubMed1994

Comparison of H5, S6, and H5-S6 exchanges on pore properties of voltage-dependent K+ channels.

Evidence is now substantial that membrane segments besides H5 contribute to the pore of K+ channels. We found that substitution of the H5 region of Shaker B (ShB) with the corresponding sequence of NGK2 expressed channels which retained the single channel K+ conductance (gK+) of the host ShB channel. A reverse chimera with ShB H5 region transplanted into NGK2 also retained the gK+ of the host NGK2. Point mutations V443L+T449Y in ShB H5 converted internal tetraethylammonium (TEA) affinity to NGK2 values, and T449Y converted external TEA affinity and Rb+ conductance (gRb+) to NGK2 values. In ShB, exchanging a short stretch of 9 amino acids located just past the transmembrane segment referred to as S6, post-S6+ produced a large increase in gK+ with no effect on internal or external TEA blockade. Within S6, 3 important residues for internal TEA blockade were identified. Thus, H5 determines external TEA blockade and both H5 and S6 may determine internal TEA blockade, but neither H5 nor S6 alone restored the donor gK+. However, chimeric channels in which H5, S6, and post-S6 were exchanged transferred gK+ of NGK2 to ShB or the gK+ of ShB to NGK2. Thus, contributions from H5, S6, and its cytoplasmic extension post-S6 make the pore of voltage-dependent Shaker K+ channels a polysegmental mosaic structure.

Amino Acid Sequence↗
PubMed1994

Rescue of lethal subunits into functional K+ channels.

In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine.

Amino Acid Sequence↗
PubMed1993

Functional interactions between K+ pore residues located in different subunits.

The aqueous pore (P-region) of homotetrameric voltage-gated K+ channels has been modeled as a radially symmetrical eight-stranded antiparallel beta-barrel to which each of the four subunits contributes equally. This model has hydrogen bonding between residues located on adjacent subunits and predicts that subunit interactions might have functional consequences. Previously we have used point mutations and an electrophysiological assay to detect functional interactions between a pair of residues at positions 369 and 374 in the P-region, but we could not distinguish between intra- and intersubunit interactions. In the present paper, we present evidence for interaction across subunit boundaries after co-injecting two cRNAs encoding subunits differing from each other at either position 369 or 374. Comparison of the phenotypes of homo- and heterotetrameric channels suggests that pore residues residing in adjacent subunits form a closely packed structure which determines both ion conductance and stability of the open state of the channel. Our results are consistent with a structure in which pore residues 369 and 374 are located in close proximity on adjacent antiparallel strands to allow both intra- and intersubunit interactions.

Amino Acid Sequence↗
PubMed1993

Inactivation determined by a single site in K+ pores.

An N-terminus peptide or a C-terminus mechanism involving a single residue in transmembrane segment 6 produces inactivation in voltage-dependent K+ channels. Here we show that a single position in the pore of K+ channels can produce inactivation having characteristics distinct from either N- or C-type inactivation. In a chimeric K+ channel (CHM), the point reversion CHM V369K produced fast inactivation and CHM V369S had the additional effect of halving K+ conductance consistent with a position in the pore. The result was not restricted to CHM; mutating position 369 in the naturally occurring channel Kv2.1 also produced fast inactivation. Like N- and C-types of inactivation, pore or P-type inactivation was characterized by short bursts terminated by rapid entry into the inactivated state. Unlike C-type inactivation, in which external tetraethylammonium (TEA) produced a simple blockade that slowed inactivation and reduced currents, in P-type inactivation external TEA increased currents. Unlike N-type inactivation, internal TEA produced a simple reduction in current and K+ occupancy of the pore had no effect. External TEA was not the only cation to increase current; external K+ enhanced channel availability and recovery from inactivation. Additional features of P-type inactivation were residue-specific effects on the extent of inactivation and removal of inactivation by a point reversion at position 374, which also regulates conductance. The demonstration of P-type inactivation indicates that pore residues in K+ channels may be part of the inactivation gating machinery.

Chimera↗
PubMed1993

Regulation of K+/Rb+ selectivity and internal TEA blockade by mutations at a single site in K+ pores.

A conservative reversion at position 374 in a chimeric K+ pore, CHM, switched the preferred ionic conductance from K+ to Rb+. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K+ channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb(+)-preferring pores while channels with the substituted polar residues Thr or Ser expressed K(+)-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K+ and Rb+ ions and a site for blockade by internal TEA.

Amino Acid Sequence↗
PubMed1993

Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores.

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chimeric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not transferred with the S5-S6 linker (pore or P region). Instead, a chimera of the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A double chimera of the cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. Because 4AP block was inhibited by tetrapentylammonium, we conclude that determinants of 4AP binding lie in the S6 segment that forms the cytoplasmic vestibule of the pore and that this site may overlap a quaternary ammonium site.

4-Aminopyridine↗
PubMed1993

Histidine substitution identifies a surface position and confers Cs+ selectivity on a K+ pore.

The amino acid located at position 369 is a key determinant of the ion conduction pathway or pore of the voltage-gated K+ channels, Kv2.1 and a chimeric channel, CHM, constructed by replacing the pore region of Kv2.1 with that of Kv3.1. To determine the orientation of residue 369 with respect to the aqueous lumen of the pore, the nonpolar Ile at 369 in Kv2.1 was replaced with a basic His. This substitution produced a Cs(+)-selective channel with Cs+:K+ permeability ratio of 4 compared to 0.1 in the wild type. Block by external tetraethylammonium (TEA) was reduced about 20-fold, while block by internal TEA was unaffected. External protons and Zn2+, that are known to interact with the imidazole ring of His, blocked the mutant channel much more effectively than the wild type channel. The blockade by Zn2+ and protons was voltage-independent, and the proton blockade had a pKa of about 6.5, consistent with the pKa for His in solution. The histidyl-specific reagent diethylpyrocarbonate produced greatly exaggerated blockade of the mutated channel compared to the wild type. The residue at position 369 appears to form part of the binding site for external TEA and to influence the selectivity for monovalent cations. We suggest that the imidazole side-chain of His369 is exposed to the aqueous lumen at a surface position near the external mouth of the pore.

Animals↗
PubMed1993

Gating-dependent mechanism of 4-aminopyridine block in two related potassium channels.

4-aminopyridine (4AP) is widely used as a selective blocker of voltage-activated K+ currents in excitable membranes, but its mechanism and site of action at the molecular level are not well understood. To address this problem we have analyzed 4AP block in Kv2.1 and Kv3.1, mammalian representatives of the Drosophila Shab and Shaw subfamilies of voltage-gated K+ channels, respectively. The two channels were expressed in Xenopus oocytes and analyzed at both the macroscopic and single channel levels. Whole cell analysis showed that 4AP sensitivity of Kv3.1 was approximately 150 times greater than that of Kv2.1. Patch clamp analysis revealed that the mechanism of 4AP block in both channels was qualitatively similar. 4AP reached its blocking site via the cytoplasmic side of the channels, the ON rate for block was strongly accelerated when channels opened and the drug was trapped in closed channels. Single channel analysis showed that 4AP decreased burst duration and increased the latency-to-first-opening. These effects were found to be related, respectively to drug ON and OFF rates in the activated channel. Kv3.1's high 4AP sensitivity relative to Kv2.1 was associated with both a slower OFF rate and therefore increased stability of the blocked state, as well as a faster ON rate and therefore increased access to the binding site. Our results indicate that in both channels 4AP enters the intracellular mouth to bind to a site that is guarded by the gating mechanism. Differences in channel gating as well as differences in the structure of the intracellular mouth may be important for specifying the 4AP sensitivity in related voltage-gated K+ channels.

4-Aminopyridine↗