Abstract
An amino acid residue was found in M2 of gamma-aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha1beta(3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha(1) subunit with the mutated beta(3) subunit beta(3)L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta(3)A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.
Highlights
The GABAA1 receptors are the major inhibitory neuronal ion channels in the mammalian central nervous system
Heteromeric ␣1CH7 receptors failed to respond to GABA (Ͻ10 nA), and an apparent outward current could be measured during application of 1 mM picrotoxin alone (Fig. 2A)
A strong transient inward current was detected during washout of picrotoxin in the absence of GABA (Fig. 2A)
Summary
Amino Acid Residue Numbering—Residues are numbered according to the mature rat sequences. Oocytes were injected with 50 nl of capped, polyadenylated cRNA dissolved in 5 mM K-HEPES, pH 6.8 This solution contained the transcripts coding for the different subunits at concentrations of 75 nM. The triangular three state model in the absence of picrotoxin (O, C, R) requires six microscopic rate constants. They are calculated using the experimentally determined current decay constants from the open to the closed states (1 and 2), the equilibrium constant for the spontaneously open state, and the law of detailed balancing. The binding of picrotoxin to the receptors adds four new rate constants to the system. The kinetics of application and washout of picrotoxin is not taken into account The integration of the differential equation system was performed using the subroutines of the Matlab 5.2.0 library (MathWorks Inc.)
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