Channel opening locks agonist onto the GABA(C) receptor

Determination of the activation mechanism of neurotransmitter-operated ion channels has been hindered by a limited understanding of the relationship b etween agonist binding and the gating of the integral ion pore. Here we des cribe a [H-3]ligand binding assay that enables us to make repeated binding measurements from the same intact oocyte expressing recombinant human rho 1 GABA(C) receptors and directly correlate the binding kinetics with electro physiological measurements. We have determined an association rate for GABA of about 10(5) M(-1)s(-1); this is four orders of magnitude slower than di ffusion, indicating GABA has restricted access to its binding site. We also demonstrate that GABA dissociates at two rates. Our data are consistent wi th the faster rate being the true microscopic dissociation rate of GABA, wi th the slower rate occurring because the opening of the pore detains agonis t release.