CHAPS solubilization of a G-protein sensitive 5-HT 1A receptor from bovine hippocampus

Abstract

The binding of [ 3H] 8-OH-DPAT to membrane-bound 5-HT 1A receptors from bovine hippocampus was saturable and corresponded to a single high-affinity state. Solubilization of the bovine hippocampal membranes with 10 mM CHAPS containing 200 mM NaCl, renders a preparation which binds [ 3H] 8-OH-DPAT with high- affinity (Kd = 1.9 nM) and is guanine nucleotide sensitive and ketanserin insensitive. 50% of [ 3H] 8-OH-DPAT binding activity is solubilized. The presence of GMP-P(NH)P promotes a low-affinity (Kd = 9.6 nM) state which is characteristic of receptors coupled to G- proteins. GMP-P(NH)P markedly accelerates the dissociation [ 3H] 8-OH-DPAT from solubilized membranes while having negligible effects on association. Thus, the agonist can activate the terniary complex rather than to promote its formation. 8-OH DPAT, WB 4101 and 5- carboxamidotryptamine dose responsively inhibit soluble [ 3H] 8-OH-DPAT binding with IC 50 values of 16.1, 15.6 and 1.3 nM, respectively. The CHAPS solubilized membrane preparation retains many of the [ 3H] 8-OH-DPAT binding characteristics of the membrane bound form.