Pentobarbital Decreases the g-Aminobutyric AcidA Receptor Subunit gamma-2 Long / Short mRNA Ratio by a Mechanism Distinct from Receptor Occupation 1

Treatment with pentobarbital of primary cultured cerebellar granule cells decreased the g-aminobutyric acid, (GABA)A receptor subunit gamma-2 long/short (gamma-2L/S) mRNA ratio. A high dose of pentobarbital (500 mM) decreased the gamma2L/S ratio by 64%; the decrease was dose and time dependent and reversible. (2)-Hexobarbital (500 mM), the less potent stereoisomer for GABAA receptor activation, decreased the ratio slightly (30%) but significantly more than (1)-hexobarbital (20%). Other GABAA receptor activators had no (100 mM ethanol) or little (2 mM 5a-pregnane-3a-ol-20-one) effect on the gamma-2L/S ratio. Furthermore, picrotoxin (10 mM), which blocks the GABAand pentobarbital-activated GABAA receptor channel, neither changed the gamma-2L/S ratio nor blocked the pentobarbital-induced changes. These data suggest that barbiturates alter the gamma-2L/S mRNA ratio by a mechanism that does not require GABAA receptor activation. The gamma2L/S subunit mRNA includes an exon encoding an octapeptide that contains a protein kinase C phosphorylation consensus site. This exon-encoded peptide, occurring in the putative intracellular loop, can be phosphorylated, and in vitro, this phosphorylation has been shown to have functional consequences. This is the first report of a drug-induced alteration in receptor mRNA splicing. Furthermore, the changes in the gamma-2L/S ratio produced by pentobarbital exposure may have significant effects on the function of an important brain protein, the GABAA receptor. The family of GABAA receptors are ligand-gated chloride channels formed from combinations of five of the . 13 mammalian receptor subunits (alpha 1–6, beta 1–3, gamma 1–3, delta and rho) in pentameric arrays (Olsen and Tobin, 1990). This multiplicity of subunits provides one basis for the diverse pharmacology of the GABAA receptors (Tyndale et al., 1995b). Further diversity can result from splicing variants of individual receptor subunit mRNAs and intracellular phosphorylation of the GABAA receptor subunits (Lin et al., 1996; Moss et al., 1992b; Swope et al., 1992). We tested the chronic effects of two GABAA receptor agonists on receptor adaptation in primary cultures of cerebellar granule cells. Initially, changes in mRNA for 13 subunits were assessed after chronic treatment. The only major change observed, however, was in the ratio of the long and short forms of mRNA for the gamma-2 subunit. The gamma-2 subunit mRNA exists in two forms, a long form (gamma-2L), whose mRNA contains a 24-nt exon, and a short form (gamma-2S), which does not contain this exon (Whiting et al., 1990; Kofuji et al., 1991). The additional exon of gamma-2L encodes an octapeptide that lies within the putative cytoplasmic domain, between the third and fourth transmembrane regions (Whiting et al., 1990; Kofuji et al., 1991). The octapeptide includes a consensus phosphorylation site for PKC, with a serine in position 343. Inclusion or exclusion of this octapeptide and its phosphorylation site may alter the phosphorylation of the resulting receptor and lead to altered receptor function (Kellenberger et al., 1992; Leidenheimer et al., 1992; Lin et al., 1996; Machu et al., 1993; Moss et al., 1992a, 1992b). Compounds that alter the ratio of gamma2L/S mRNAs may also alter the functional properties of GABAA receptors; therefore, such compounds will enable further exploration of the role of receptor phosphorylation in