Open-channel Blocking Mechanism Research Articles

Novel, Potent Inhibitors of Human Kv1.5 K+ Channels and Ultrarapidly Activating Delayed Rectifier Potassium Current

We have identified a series of diphenyl phosphine oxide (DPO) compounds that are potent frequency-dependent inhibitors of cloned human Kv1.5 (hKv1.5) channels. DPO inhibited hKv1.5 expressed in Chinese hamster ovary cells in a concentration-dependent manner preferentially during channel activation and slowed the deactivating tail current, consistent with a predominant open-channel blocking mechanism. Varying kinetics of DPO interaction with Kv1.5 channels resulted in differing potencies and frequency dependencies of inhibition that were comparable for both expressed hKv1.5 current and native ultrarapidly activating delayed rectifier potassium current (IKur) in human atrial myocytes. Selectivity of DPO versus other cardiac K+ channels was demonstrated in human atrial myocytes (IKur versus transient outward potassium current) and guinea pig ventricular myocytes [IKur versus rapidly activating delayed rectifier potassium current (IKr), slowly activating delayed rectifier potassium current (IKs) and inward rectifier potassium current (IK1), and one compound (DPO-1) was shown to be 15-fold more selective for Kv1.5 versus Kv3.1 channels expressed in Xenopus oocytes. DPO-1 also prolonged action potentials of isolated human atrial but not ventricular myocytes, in contrast to the effect of a selective IKr blocker. The selectivity and kinetics of inhibition hKv1.5 and IKur by DPO and the resulting selective prolongation of atrial repolarization could provide an effective profile for treatment of supraventricular arrhythmias.

Flecainide sensitivity of a Na channel long QT mutation shows an open-channel blocking mechanism for use-dependent block

A long QT mutation in the cardiac sodium channel, D1790G (DG), shows enhanced flecainide use-dependent block (UDB). The relative importance of open and inactivated states of the channel in flecainide UDB has been controversial. We used a modifiable, inactivation-deficient mutant channel that contains the F1486C mutation in the IFM motif to investigate the UDB difference between the wild-type (WT-ICM) and DG (DG-ICM) channels. UDB at 5 Hz was greater in DG-ICM than WT-ICM, and IC50 values for steady-state UDB were 7.19 and 18.06 microM, respectively. When [2-(trimethyammonium) ethyl]methanethiosulfonate bromide (MTSET) was included in the pipette and fast inactivation was disabled, IC50 was 5.04 microM for DG-ICM and 12.63 microM for WT-ICM. We measured open-channel block by flecainide directly in MTSET-treated, noninactivating ICM channels. Steady-state block was higher for DG-ICM than WT-ICM (IC50 was 2.34 microM for DG-ICM and 5.87 microM for WT-ICM), suggesting that open-channel block is an important determinant of flecainide UDB. We obtained association (kon) and dissociation (koff) rates for open-channel block by the Langmuir-isotherm model. They were koff = 31.37 s(-1), kon = 5.83 s(-1).microM(-1), and calculated Kd = 5.38 microM for WT-ICM (where Kd = koff/kon); and koff = 24.88 s(-1), kon = 9.54 s(-1).microM(-1), and calculated Kd = 2.61 microM for DG-ICM. These Kd values were similar to IC50 measured from steady-state open-channel block. Furthermore, we modeled UDB mathematically by using these kinetic rates and found that the model predicted experimental UDB accurately. The recovery from UDB had a minor contribution to UDB. Flecainide UDB is predominantly determined by an open-channel blocking mechanism, and DG-ICM channels appeared to have an altered open-channel state with higher flecainide affinity than WT-ICM.

Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.

The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.

The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano-electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage-dependent blocking kinetics indicate an open-channel block mechanism, which can be well described by a two barrier-one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca2+ concentration in the endolymph surrounding the hair bundles is < 100 microM, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.

Two mechanisms of action of the adamantane derivative IEM‐1460 at human AMPA‐type glutamate receptors

1. Antagonizing glutamatergic neurotransmission by blockade of AMPA-type glutamate receptors (GluR) is a promising pharmacological strategy for neuroprotection in neurodegenerative diseases and acute treatment of stroke. 2. We investigated the interaction of the adamantane derivative IEM-1460 with human wild-type and mutant AMPA-type GluR channels. Different recombinant homooligomeric human AMPA-type GluR channels and a rat nondesensitizing mutant GluR (GluR2 L504Y) channel were expressed in HEK293 cells and investigated using the patch-clamp technique in combination with ultrafast agonist application. 3. When IEM-1460 was coapplied with glutamate, an open channel block mechanism was observed at slow desensitizing GluR2 flip (>/=0.1 mM IEM-1460) and nondesensitizing GluR2 L504Y channels (>/=1 microM IEM-1460). 4. A competitive block of AMPA-type channels was observed with IC(50) values for the dose block curves of 0.1 mM IEM-1460 at human unmutated and 10 microM IEM-1460 at mutant GluR channels. 5. Nondesensitizing GluR2 L504Y channels were used to further characterize the block mechanism. After equilibration with the agonist, a current decay upon coapplication of glutamate and IEM-1460 was observed. The recovery from block was independent of the glutamate and IEM-1460 concentration. The extent of current inhibition as well as the time constant of current decay upon addition of the blocker to the test solution were dependent on agonist concentration; this strongly points to an additional competitive-like block mechanism of IEM-1460 at human AMPA-type GluR channels. 6. The data were interpreted in the frame of a molecular scheme with two binding sites of IEM-1460 at the receptor, one at the unliganded resting and the other at the fully liganded open state of the channels.

Comments on “Memantine Blocks α7* Nicotinic Acetylcholine Receptors More Potently Than N-Methyl-d-aspartate Receptors in Rat Hippocampal Neurons”

The N-methyl-d-aspartate (NMDA) receptor antagonist memantine is an approved drug for treatment of Alzheimer's disease (AD). Other such treatments are cholinesterase inhibitors and nicotinic acetylcholine receptor (nAChR)-sensitizing agents such as galantamine. The present study was designed to test whether memantine exerts any effect on the cholinergic system, in particular the Ca(2+)-conducting alpha7(*) nAChR, in cultured hippocampal neurons. Memantine caused a concentration-dependent reduction of the amplitudes of whole-cell currents evoked by the alpha7(*) nAChR-selective agonist choline (10 mM) or by N-methyl-d-aspartate (NMDA) (50 muM) plus glycine (10 muM). It also inhibited tonically activated NMDA receptors. Memantine was more potent in inhibiting alpha7(*) nAChRs than NMDA receptors; at -60 mV, the IC(50) values for memantine were 0.34 and 5.1 muM, respectively. Consistent with an open-channel blocking mechanism, memantine-induced NMDA receptor inhibition was voltage and use-dependent; the Hill coefficient (n(H)) was approximately 1. Memantine-induced alpha7(*) nAChR inhibition had an n(H) < 1 and showed a variable voltage dependence; the effect was voltage-independent at 0.1 muM, becoming voltage-dependent at >/=1 muM. Thus, memantine interacts with more than one class of sites on the alpha7(*) nAChRs. One is voltage-sensitive and therefore likely to be within the receptor channel. The other is voltage-insensitive and therefore likely to be in the extracellular domain of the receptor. It is suggested that blockade of alpha7(*) nAChRs by memantine could decrease its effectiveness for treatment of AD, particularly at early stages when the degrees of nAChR dysfunction and of cognitive decline correlate well.

Memantine Blocks α7*Nicotinic Acetylcholine Receptors More Potently ThanN-Methyl-D-aspartate Receptors in Rat Hippocampal Neurons

The N -methyl-d-aspartate (NMDA) receptor antagonist memantine is an approved drug for treatment of Alzheimer's disease (AD). Other such treatments are cholinesterase inhibitors and nicotinic acetylcholine receptor (nAChR)-sensitizing agents such as galantamine. The present study was designed to test whether memantine exerts any effect on the cholinergic system, in particular the Ca2+-conducting α7* nAChR, in cultured hippocampal neurons. Memantine caused a concentration-dependent reduction of the amplitudes of whole-cell currents evoked by the α7* nAChR-selective agonist choline (10 mM) or by N -methyl-d-aspartate (NMDA) (50 μM) plus glycine (10 μM). It also inhibited tonically activated NMDA receptors. Memantine was more potent in inhibiting α7* nAChRs than NMDA receptors; at -60 mV, the IC50 values for memantine were 0.34 and 5.1 μM, respectively. Consistent with an open-channel blocking mechanism, memantine-induced NMDA receptor inhibition was voltage and use-dependent; the Hill coefficient ( n H) was ∼1. Memantine-induced α7* nAChR inhibition had an n H < 1 and showed a variable voltage dependence; the effect was voltage-independent at 0.1 μM, becoming voltage-dependent at ≥1 μM. Thus, memantine interacts with more than one class of sites on the α7* nAChRs. One is voltage-sensitive and therefore likely to be within the receptor channel. The other is voltage-insensitive and therefore likely to be in the extracellular domain of the receptor. It is suggested that blockade of α7* nAChRs by memantine could decrease its effectiveness for treatment of AD, particularly at early stages when the degrees of nAChR dysfunction and of cognitive decline correlate well.

Steady-state interactions of glibenclamide with CFTR: evidence for multiple sites in the pore.

The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.

Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation

In HEK293 cells stably expressing α4β2 nAChRs, naltrexone, but not naloxone, blocked α4β2 nAChRs via an open-channel blocking mechanism. In primary hippocampal cultures, naltrexone inhibited α7 nAChRs up-regulated by nicotine, and in organotypic hippocampal cultures naltrexone caused a time-dependent up-regulation of functional α7 nAChRs that was detected after removal of the drug. These results indicate that naltrexone could be used as a smoking cessation aid.

Inhibition of ultra-rapid delayed rectifier K + current by verapamil in human atrial myocytes

Verapamil is a widely used Ca 2+ channel antagonist in the treatment of cardiovascular disorders including atrial arrhythmias. However, it is unknown whether the drug would inhibit the repolarization currents transient outward K + current ( I to1) and ultra-rapid delayed rectifier K + current ( I Kur) in human atrium. With whole-cell patch configuration, we evaluated effects of verapamil on I to1 and I Kur in isolated human atrial myocytes. It was found that verapamil did not decrease I to1 at 1–50 μM. However, verapamil reversibly inhibited I Kur in a concentration-dependent manner (IC 50 = 3.2 μM). At test potential of +50 mV, 5 μM verapamil decreased I Kur by 61.3 ± 7.5%. Verapamil significantly accelerated inactivation of I Kur, suggesting an open channel block mechanism. The results indicate that verapamil significantly blocks the repolarization K + current I Kur, but not I to1, in human atrial atrium, which may account at least in part for the atrial effect of the drug.

Activation and block of mouse muscle-type nicotinic receptors by tetraethylammonium

We have studied the activation and inhibition of the mouse muscle adult-type nicotinic acetylcholine receptor by tetraethylammonium (TEA) and related quaternary ammonium derivatives. The data show that TEA is a weak agonist of the nicotinic receptor. No single-channel clusters were observed at concentrations as high as 5 mM TEA or in the presence of a mutation which selectively increases the efficacy of the receptor. When coapplied with 1 mM carbamylcholine (CCh), TEA decreased the effective opening rate demonstrating that it acts as a competitive antagonist of CCh-mediated activation. Kinetic analysis of currents elicited by CCh and TEA allowed an estimate of receptor affinity for TEA of about 1 mM, while an upper limit of 10 s−1 could be set for the wild-type channel-opening rate constant for receptors activated by TEA alone. At millimolar concentrations, TEA inhibited nicotinic receptor currents by depressing the single-channel amplitude. The effect had an IC50 of 2–3 mM, depending on the conditions of the experiment, and resembled a standard open-channel block. However, the decrease in channel amplitudes was not accompanied by an increase in the mean burst duration, indicating that a linear open-channel blocking mechanism is not applicable. Upon studying block by other nicotinic receptor ligands it was found that block by CCh, tetramethylammonium and phenyltrimethylammonium can be accounted for by the sequential blocking mechanism while block in the presence of methyltriethylammonium, ethyltrimethylammonium or choline was inconsistent with such a mechanism. A mechanism in which receptors blocked by TEA can close would account for the experimental findings.

Cu2+-induced modification of the kinetics of A beta(1-42) channels.

We found that the amyloid beta peptide A beta(1-42) is capable of interacting with membrane and forming heterogeneous ion channels in the absence of any added Cu2+ or biological redox agents that have been reported to mediate A beta(1-42) toxicity. The A beta(1-42)-formed cation channel was inhibited by Cu2+ in cis solution ([Cu2+]cis) in a voltage- and concentration-dependent manner between 0 and 250 microM. The [Cu2+]cis-induced channel inhibition is fully reversible at low concentrations between 50 and 100 microM [Cu2+]cis and partially reversible at 250 microM [Cu2+]cis. The inhibitory effects of [Cu2+]cis between 50 and 250 microM on the channel could not be reversed with addition of Cu2+-chelating agent clioquinol (CQ) at concentrations between 64 and 384 microM applied to the cis chamber. The effects of 200-250 microM [Cu2+]cis on the burst and intraburst kinetic parameters were not fully reversible with either wash or 128 microM [CQ]cis. The kinetic analysis of the data indicate that Cu2+-induced inhibition was mediated via both desensitization and an open channel block mechanism and that Cu2+ binds to the histidine residues located at the mouth of the channel. It is proposed that the Cu2+-binding site of the A beta(1-42)-formed channels is modulated with Cu2+ in a similar way to those of channels formed with the prion protein fragment PrP(106-126), suggesting a possible common mechanism for Cu2+ modulation of A beta and PrP channel proteins linked to neurodegenerative diseases.

Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241.

1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC(50) of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-specific. 3 The block of hEAG1 channels by LY97241 and clofilium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC(50) of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on-rate of block. 6 Physico-chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.

The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site.

Transient outward current inhibition by propafenone and 5-hydroxypropafenone in cultured neonatal rat ventricular myocytes.

The antiarrhythmic agent propafenone and its primary electropharmacologically active metabolite, 5-hydroxypropafenone, are known inhibitors of cardiac myocyte repolarizing currents. We recently documented potent propafenone inhibition of the transient outward potassium current (Ito) in human atrial myocytes from patients in the newborn and infant age range. In the current study we characterized ventricular Ito inhibition by propafenone and 5-hydroxypropafenone in neonatal myocytes enzymatically isolated from 2-day-old Sprague-Dawley rat pups. Using the whole-cell patch-clamp technique in ventricular myocytes kept in primary culture for 1-4 days, we observed comparably potent Ito inhibition by both agents, yielding 50% maximal inhibitory concentration (IC50) values of 2.1 +/- 0.5 and 1.5 +/- 0.2 microM for propafenone and 5-hydroxypropafenone, respectively. Ito blockade by both of these agents was time, concentration, and voltage dependent, but use independent. There was no drug effect on steady-state voltage dependence of Ito inactivation, or on the time course of Ito recovery from inactivation. These findings are consistent with an open channel-blocking mechanism as suggested by other models. We conclude that both propafenone and 5-hydroxypropafenone are potent Ito inhibitors in neonatal rat ventricular myocytes, with potencies exceeding those demonstrated for propafenone in adult rat ventricular myocytes or in human atrial myocytes from patients of all ages.

Testosterone-mediated modulation of HERG blockade by proarrhythmic agents

Diverse drugs from many therapeutic classes exert cardiotoxic side effects by inducing torsades de pointes (TdP), a life threatening cardiac arrhythmia, which often results from drug interaction with HERG (human ether-a-go-go related gene) encoded K + channels, that generate an I Kr component of the delayed rectifier cardiac K + current. Men are known to be at a lower risk for drug-induced TdP than women suggesting a role of sex steroid hormones, androgens and estrogens, in modulation of drug sensitivity of cardiac K + channels, particularly those encoded by HERG. Here by using neuroleptic agents haloperidol, pimozide, and fluspirilene, all of which can induce TdP, and a steroid hormone-sensitive system Xenopus oocytes for HERG channels expression we show that testosterone is able to reduce HERG-blocking potency of neuroleptics. Haloperidol, pimozide, and fluspirilene inhibited HERG current with ic 50 of 1.36, 1.74, and 2.34 μM, and maximal block of 73%, 76% and 65%, respectively. The action of these neuroleptics was voltage-dependent, most consistent with an open-channel blocking mechanism. Pretreatment of HERG-expressing oocytes with 1 μM testosterone increased the ic 50 values to 2.73, 2.08, and 5.04 μM, reduced the maximal block to 65%, 59%, and 64%, and strongly diminished voltage-dependence of the blockade. Testosterone treatment per se produced about a 35% reduction of HERG current compared with untreated oocytes. Our data suggest that androgens may protect against the arrhythmogenic actions of some cardiotoxic drugs.

Verapamil block of large-conductance Ca-activated K channels in rat aortic myocytes.

The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state-specifically, a nonconducting, open-blocked state--was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.

The n-alcohol site in the nicotinic receptor pore is a hydrophobic patch.

Alcohols and volatile anesthetics inhibit peripheral nicotinic acetylcholine receptors noncompetitively, primarily via an open-channel block mechanism. Analysis of hydrophobic mutations near the middle of the pore-forming M2 domains suggested that alcohols interact with the pore in this vicinity. To establish the extent of this inhibitory site, we created a series of hydrophobicity-altering mutations scanning most of the alpha subunit M2 domain. Using both single-channel and rapid patch perfusion electrophysiology, we measured how these mutations affect nAChR sensitivity to ethanol and hexanol. We find a near-contiguous series of amino acids in alpha-M2, extending from alphaL250 (8') to alphaV255 (13'), where mutagenesis strongly influences inhibition by alcohols. These results support the existence of a large inhibitory patch in the nAChR pore lining where interactions with alcohols are primarily due to hydrophobic forces. Ethanol appears to interact with deeper regions of this site than does hexanol. Because alcohols apparently act as open-channel blockers, we infer from our results that most of the residues between alphaL250 and alphaV255 are exposed to the aqueous environment of the pore when the channel is open. The location and extent of this site can explain why small alcohols occupy the nAChR pore at the same time as larger alcohols or charged blockers, while two large alcohols bind in a mutually exclusive manner.

Modulation by bicuculline and penicillin of the block by t-butyl-bicyclo-phosphorothionate (TBPS) of GABA(A)-receptor mediated Cl(-)-current responses in rat striatal neurones.

1. T-butyl-bicyclo-phosphorothionate (TBPS) is a prototypical representative of the cage-convulsants which act through a use-dependent block of the GABA(A)-receptor-ionophore complex. Using current recordings from cultured neurones of rat striatum the manner was investigated in which two antagonists, bicuculline and penicillin, presumably acting at the agonist binding site and in the ionic channel, respectively, modify the rate of block by TBPS. 2. Penicillin (5 or 10 mM) did not slow the rate of block by TBPS, but produced a significant enhancement of block rate, which, however, was inversely related to the degree of antagonism by penicillin of the GABA-induced current. 3. Bicuculline (10 microM) reduced the rate of block by TBPS. However, this effect was 3 fold weaker than its GABA-antagonistic action. The slowing of block rate and the current antagonism exhibited a biphasic, positive-negative relationship. Co-application of bicuculline (100 microM) in a concentration that produced nearly complete antagonism and TBPS (10 microM) resulted in a marked ( approximately 40%) reduction of subsequent GABA response amplitudes compatible with a direct, bicuculline-induced conformational change in the receptor required for the binding of and block by TBPS. 4. The lack of protection afforded by the channel blocker penicillin as well as the lack of correlation between bicuculline antagonism of the Cl(-)-current and its efficiency in protecting against TBPS block is evidence against an open channel blocking mechanism for TBPS. TBPS does, therefore, not appear to gain access to its binding site via the open pore but through alternative routes regulated from the agonist binding site.

Non‐specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2

The alpha1-adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred microM methoxamine reduced Ito by 36% in the presence of 2 microM prazosin, and by 37 and 38% after preincubation of myocytes with either N-ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 microM, respectively, with Hill coefficients of 0.87-1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration- and voltage-dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose-response relations. The voltage-dependence of block supported charged methoxamine binding to a putative intracellular site that sensed approximately 20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single-channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 microM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine.