DEVELOPMENTAL EXPRESSION OF FUNCTIONAL GABA(A) RECEPTORS CONTAINING THE GAMMA-2 SUBUNIT IN NEURONS DERIVED FROM EMBRYONAL CARCINOMA (P19) CELLS

The expression of the gamma 2 subunit into functional GABA(A) receptor s has been examined in the embryonal carcinoma (EC) cell line P19, a p luripotent cell line which differentiates into a neuronal phenotype af ter exposure to retinoic acid. Whole-cell voltage-clamp recordings wer e used to examine the characteristics of the GABA receptors expressed in P19 cells at different times after exposure to retinoic acid. Messe nger RNA for both the gamma 2L and gamma 2S splice variants of the GAB A(A) receptor increased dramatically following differentiation of P19 EC cells with retinoic acid. By 12 days after retinoic acid treatment, while both mRNAs were present, there was an approximately 10-fold gre ater abundance of gamma 2S mRNA compared to gamma 2L. However, at this same time point neurons derived from P19 cells stained intensely with a polyclonal antibody raised against a peptide fragment specific for the gamma 2L subunit. A significant increase in both the affinity for GABA and the maximum current amplitude elicited by GABA occurred betwe en 7 and 12 days after retinoic acid treatment. In contrast, the abili ty of the benzodiazepine agonist flurazepam to potentiate GABA-induced membrane current was the same at 7 and 12 days after retinoic acid tr eatment. These data suggest that the gamma 2 subunit of the GABA(A) re ceptor is expressed early following differentation of P19 cells into a neuronal phenotype, and that this subunit is incorporated into functi onal GABA(A) receptors. Moreover, the gamma 2S and gamma 2L splice var iants of this subunit may be co-expressed in neurons derived from P19 cells. The observed affinity change for GABA may reflect a time-depend ent change in the expression of alpha and/or beta subunits of the GABA (A) receptor, as occurs in developing neuronal tissue both in vitro an d in vivo.