Few cell lines with GABAA mRNAs have functional receptors

Abstract

In the preceding companion article (Tyndale et al., 1994) we used the PCR to investigate the occurrence of 13 GABAA receptor subunit mRNAs in several cell lines, including those derived from brain (B65, B104, and NB41A3), cerebellum (C17), glia (C6), pituitary (AtT-20), adrenal medulla (PC12), and the endocrine pancreas (RINmSF and βTC3). In the present study we used the whole-cell configuration of the patch-clamp technique to determine which of these cell lines express functional GABAA receptors. All of the cell lines contain detectable levels of at least one GABAA receptor subunit mRNA (Tyndale et al., 1994); however, only RINm5F and βTC3 cells exhibited GABA-evoked currents. GABA activated currents in all RINm5F cells, but currents were only barely detectable in 50% of βTC3 cells tested. Many of the cell lines that failed to respond to GABA were derived from cell types with functional GABAA receptors. For example, the failure of PC12 cells to respond to GABA contrasts with the observation of GABA responses recorded from all primary cultured adrenomedullary chromaffin cells tested. GABA-evoked currents recorded from βTC3 cells were too small (< 10 pA) to characterize pharmacologically. However, GABA activated robust currents recorded from RINm5F cells. These currents reversed at a holding potential similar to the equilibrium potential for Cl-, were blocked by the antagonist bicuculline methiodide (10 μM), and were potentiated by pentobarbital (100 μM). RINm5F cell GABAA receptors were insensitive to diazepam (10 μM) and were inhibited by Zn2+ (10 μM). For comparison, diazepam (5 μM) caused a large potentiation and Zn2+ (100 μM) only a relatively small inhibition of GABA responses recorded from primary cultured chromaffin cells. Benzodiazepine modulation is dependent on the γ GABAA receptor subunit and Zn2+ inhibits only those receptors lacking the subunit. Hence, taken together these observations suggest that in contrast to chromaffin cells, RINm5F cells express functional GABAA receptors that lack γ subunits. Many neuronal and peripheral cell lines express GABAA receptor mRNAs, but few have functional receptors. The occurrence of functional GABAA receptors in cell lines is discussed in relation to their tissues of origin.