Molecular pharmacology
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Molecular pharmacology · Aug 2000
Comparative StudySteroid inhibition of rat neuronal nicotinic alpha4beta2 receptors expressed in HEK 293 cells.
Steroids, in addition to regulating gene expression, directly affect a variety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha4beta2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine responses from these receptors, with slow kinetics requiring seconds for block to develop and recover. ⋯ The stereochemistry at the 3 and 5 positions was less influential for block of alpha4beta2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of steroids to block neuronal nicotinic receptors correlated with their ability to produce anesthesia in Xenopus tadpoles, but the concentrations required for inhibition are generally greater. Similarly, the concentrations of endogenous neurosteroids required to inhibit receptors are larger than estimates of brain concentrations.
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Molecular pharmacology · Aug 2000
Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine.
Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. ⋯ In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.
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Molecular pharmacology · Mar 2000
A single glycine residue at the entrance to the first membrane-spanning domain of the gamma-aminobutyric acid type A receptor beta(2) subunit affects allosteric sensitivity to GABA and anesthetics.
Site-directed mutagenesis of the gamma-aminobutyric acid type A (GABA(A)) receptor beta(2) subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous rho(1) residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [(3)H]muscimol and [(3)H]flunitrazepam binding in receptors containing the beta(2)(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA(A) receptors containing the alpha(1)(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. ⋯ Although ligand binding displayed comparable K(D) values for muscimol among wild-type, alpha(1)beta(2)gamma(2), and mutant receptors, patch-clamp recordings showed that alpha(1)beta(2)(G219F)gamma(2) receptors had a significantly more potent response to GABA than did alpha(1)beta(2)gamma(2) or alpha(1)(G223F)beta(2)gamma(2). The alpha(1)beta(2)(G219F)gamma(2) receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the beta(2) subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.
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Molecular pharmacology · Dec 1999
Putative partial agonist 1-aminocyclopropanecarboxylic acid acts concurrently as a glycine-site agonist and a glutamate-site antagonist at N-methyl-D-aspartate receptors.
1-Aminocyclopropanecarboxylic acid (ACPC) has been shown to protect against neuronal cell death after ischemic insult in vivo. Such results can be correlated with in vitro assays in which ACPC protected neurons against glutamate-induced neurotoxicity by reducing the activity of N-methyl-D-aspartate (NMDA) channel activation. Electrophysiological studies have determined that ACPC inhibits NMDA receptor activity by acting as a glycine-binding site partial agonist. ⋯ The removal of ACPC initially caused an increase in inward current followed by a subsequent decrease to baseline levels. This suggests that relief of low-affinity antagonism occurs before high-affinity agonist dissociation. Simulations of ACPC action by a two glutamate-binding site/two glycine-binding site model for NMDA channel activation in conjunction with the concurrent role of ACPC as a glycine-site full agonist and glutamate-site competitive antagonist were able to successfully approximate experimental results.
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Molecular pharmacology · Dec 1999
Comparative StudyDifferential interaction of R-mexiletine with the local anesthetic receptor site on brain and heart sodium channel alpha-subunits.
Mexiletine is a class I antiarrhythmic drug with neuroprotective effects in models of brain ischemia attributable to inhibition of brain sodium channels. We compared effects of R-mexiletine on wild-type and mutant rat brain (rbIIA) and heart (rh1) sodium channel alpha-subunits transiently expressed in tsA-201 cells. R-mexiletine induced tonic and frequency-dependent block and bound with a 26-fold (brain) or 35-fold (heart) higher affinity to inactivated sodium channels. ⋯ Unlike previous local anesthetics studied, the strongest effect was observed for mutation rbY1771A. Comparison of mutations of the homologous phenylalanine residue in brain and heart channels showed striking differences in the effects of the mutations. rbF1764A reduced drug block by slowing R-mexiletine binding to inactivated channels, whereas rhF1762A reduced block by increasing the rate of dissociation from inactivated and resting channels. Thus, rbF1764/rhF1762 is a critical determinant of affinity and tissue-specific differences in mexiletine block of brain and heart sodium channels, but its role in drug interaction differs in these two channel isoforms.