Electrophysiological and binding studies of kainate receptor-channels in mammalian brain.

摘要

This dissertation focuses on the pharmacological and biophysical characterization of the kainate receptor binding sites and the ionic channels through which kainate mediates its responses to the mammalian cerebellar and cortical neurons. Particular attention was paid to the long standing question of whether the non-NMDA agonists AMPA and kainate activate separate receptor-channels with different conductance states or a single channel population that has complex kinetics and agonist dependent open state behavior.; ({dollar}sp3{dollar}H) kainate saturation binding assays on crude synaptosomal membranes from rabbit cerebellum revealed at least two independent binding sites with a 10-fold difference in affinity (K{dollar}sb{lcub}rm dhigh{rcub}{dollar} = 1.5-2.0, and K{dollar}sb{lcub}rm dlow{rcub}{dollar} = 17-20 nM) for the radioligand. These sites were pharmacologically distinct based upon their different sensitivity to AMPA, ibotenic acid, and cGMP as revealed from competition assays.; The effects of the compounds above were compared to the actions of kainate in electrophysiological studies carried out in whole cell and excised patch recordings from cerebellar and cortical neurons where macroscopic currents were obtained and subjected to noise analysis. Single channel current events were examined in detail in outside-out patches.; Slow bath application of kainate activated current responses that could be classified into three main groups based on the estimated conductances: the low conductance channels (0.5-3 pS), the intermediate conductance channels (12-15 and 22-26 pS) and the large conductance channels (30-40 pS). The low conductance channel group showed the largest heterogeneity with respect to their pharmacology and kinetic profile. AMPA was found to activate the intermediate and larger conductance channels when applied to the same cells or patches as kainate. However, slow bath application of AMPA never activated low conductance currents, while some low conductance kainate responses were inhibited following co-application of AMPA with kainate. From these results it was concluded that both AMPA and kainate interact with receptors coupled to intermediate and low conductance channels; however the functional consequences of the interactions differ possibly accounting for some of the controversy of whether or not there are separate kainate and AMPA receptors.; In studies designed to determine whether or not the apparent nonlinear Rosenthal plots of specific ({dollar}sp3{dollar}H) kainate binding could be accounted for by the involvement of a G-protein in the receptor-effector system, it was discovered quite unexpectedly that guanosine nucleotides including cGMP and GTP were equipotent displacers of the ({dollar}sp3{dollar}H) kainate binding in competition assays. Adenosine analogs had no effect. Following up this observation with electrophysiological studies it was shown that the guanosine compounds act on the extracellular domain of the receptor most probably as competitive antagonists of the kainate and AMPA responses mediated by the intermediate conductance receptors.