The binding isotherm of the agonist [<sup>3</sup>H]muscimol to the α4β3N-Flag-δ GABA<sub>A</sub> receptor in native membranes and reconstituted into CHAPS/lipid micelles.

Abstract

<p>Binding curves of [<sup>3</sup>H]muscimol to α4β3N–Flag–δ GABA<sub>Α</sub>Rs, both, in cell membranes (pmol/mg membrane protein) and after purification and reconstitution into micelles of 5 mM CHAPS and 200 μM DOPC:DOPS:Cholesterol in mole ratio 52:15:33 (pmol/mL). Displaceable binding was determined as the difference between binding in the presence and absence of 1 mM GABA using a filtration assay in triplicate. The displaceable binding and its standard deviation was determined by subtracting these two values and propagating errors at each total muscimol concentration. The curves were fitted by nonlinear least squares with weighting by standard deviation. These yielded apparent dissociation constants of 9.2 ± 0.6 and 35 ± 12 nM, respectively. The B<sub>max</sub> of the membranes was 22.6 ± 0.5 pmol/mg and for reconstituted receptors in micelles was 19 ± 3 pmol/mL. The Hill coefficients differed little from one (1.07 ± 0.3 and 0.90 ± 0.05 respectively).</p

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