1ST DIRECT ELECTRON-MICROSCOPIC VISUALIZATION OF A TIGHT SPATIAL COUPLING BETWEEN GABA-A-RECEPTORS AND VOLTAGE-SENSITIVE CALCIUM CHANNELS

Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect immunogold labelling using a monoclonal antibody specific for phenylalkylamine and dihydropyridine sensitive Ca2+ channels. Using the same technique and a monoclonal antibody (bd-17) to the beta(2)/beta(3)-subunit of the GABA(A)-receptor, double labelling of Ca2+ channels and GABA(A)-receptors with gold particles of different and well defined sizes were performed. This showed that in THIP-treated cultures 20% of GABA(A)-receptors in cell processes were located in close proximity (i.e. within 40 nm) of Ca2+ channels in the plasma membrane. This was not observed in non-treated cultures nor was it observed in cell bodies of THIP-treated cultures. This suggests that primarily low affinity GABA(A)-receptors are closely associated with Ca2+ channels and this may be important for the ability of these receptors to mediate an inhibitory action on transmitter release even under extreme depolarizing conditions.