High- and low-affinity GABA-receptors in cultured cerebellar granule cells regulate transmitter release by different mechanisms

The ability of high- and low-affinity GABA_A-receptors, respectively to inhibit depolarization coupled transmitter release was studied in cultured glutamatergic cerebellar granule cells which, depending on the culture conditions, express either high-affinity GABA_A-receptors alone or high-affinity receptors together with low-affinity receptors. In order to gain information about the coupling of these receptors to chloride channels the effect of picrotoxin and binding of ³⁵S]t-butylbicyclophosphorothionate, both of which interact specifically with such channels were studied. Moreover, the influence of Flunitrazepam on the GABA-mediated inhibition of transmitter release was investigated to see if the GABA-receptors are coupled to benzodiazepine binding sites. Under conditions where the granule cells express only high-affinity GABA_A-receptors it was found that GABA was able to inhibit transmitter release elicited by mild depolarization induced either by 30 mM KCl or 25 μM glutamate. This effect of GABA could be enhanced by Flunitrazepam and blocked by picrotoxin. However, transmitter release from these neurones induced by a more pronounced depolarization (55 mM KCl) could not be inhibited by GABA. Under conditions where the neurons express both high- and low-affinity GABA_A-receptors transmitter release elicited by 55 mM KCl could be inhibited by GABA but this inhibitory effect of GABA could not be blocked by picrotoxin, nor could it be enhanced by Flunitrazepam. These results strongly suggest that while the action of the high-affinity GABA_A-receptors is coupled to chloride channels and benzodiazepine binding sites, the physiological action of the low-affinity GABA_A-receptors is not. This lack of coupling between the low-affinity GABA_A-receptors and chloride channels is further supported by the finding that the K_D and B_max values for ³⁵S]TBPS binding to the granule cells were independent of whether or not the cells expressed low-affinity GABA_A-receptors. While the results clearly show that the inhibitory action of GABA mediated by low-affinity GABA_A-receptors is not coupled to chloride channels, the exact mechanism of action of these receptors still remains to be elucidated.