Molecular pharmacology
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Molecular pharmacology · Apr 2001
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. ⋯ 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.
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Molecular pharmacology · Apr 2001
Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by gamma-aminobutyric acid(A) receptors in hippocampal neurons.
gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABA(A) receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. ⋯ Furthermore, compounds that potentiate GABA(A) receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinically-relevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs.
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Molecular pharmacology · Feb 2001
Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function.
Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis oocytes expressing endogenous LPA and trypsin or recombinant m1 receptors were two-electrode voltage clamped. ⋯ Lidocaine and its analog QX314 were injected into oocytes expressing these receptors to examine a potential role for specific G protein alpha-subunits as targets for LA. Galpha(q) was shown to be among the primary G protein subunits mediating the LPA, m1, and trypsin receptor signaling, all of which were inhibited to a similar degree by intracellular injected QX314 (424 x 10(-6) M). Since the angiotensin(1A) receptor, previously shown not to be affected by LA, was found not to signal via Galpha(q), but via Galpha(o) and Galpha(14), the intracellular effect of LA most likely takes place at the Galpha(q)-subunit.
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Molecular pharmacology · Feb 2001
Irreversible block of human heart (hH1) sodium channels by the plant alkaloid lappaconitine.
The roots from Aconitum sp. plants have long been used in Chinese herbal medicine for treating pain and various heart conditions. The principal component of Aconitum remedies is usually aconitine, a site 2 neurotoxin that may induce severe neurological symptoms and cardiovascular collapse. Some Aconitum species also contain lappaconitine, the structure of which is remarkably similar to that of aconitine. ⋯ Whereas site 2 neurotoxins often irreversibly modify channel kinetics, lappaconitine irreversibly blocks the channels. Finally, channels containing lysine substitutions within the local anesthetic receptor region at residues F1760 or N1765 are resistant to block by bupivacaine or lappaconitine. Given that site 2 neurotoxins and local anesthetics have nonidentical but overlapping binding regions, these data suggest that lappaconitine irreversibly blocks hH1 channels by binding to the site 2 receptor.
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Molecular pharmacology · Sep 2000
Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels.
Retigabine [N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid; D-23129] is a novel anticonvulsant, unrelated to currently available antiepileptic agents, with activity in a broad range of seizure models. In the present study, we sought to determine whether retigabine could enhance current through M-like currents in PC12 cells and KCNQ2/Q3 K(+) channels expressed in Chinese hamster ovary cells (CHO-KCNQ2/Q3). In differentiated PC12 cells, retigabine enhanced a linopirdine-sensitive current. ⋯ Retigabine shifted the voltage dependence of channel activation with an EC(50) value of 1.6 +/- 0.3 microM (slope factor was 1.2 +/- 0.1, n = 4 to 5 cells per concentration). Retigabine (0.1 to 10 microM) also slowed the rate of channel deactivation, predominantly by increasing the contribution of a slowly deactivating tail current component. Our findings identify KCNQ2/Q3 channels as a molecular target for retigabine and suggest that activation of KCNQ2/Q3 channels may be responsible for at least some of the anticonvulsant activity of this agent.