Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons

Abstract

Rohon-Beard (RB) spinal neurons of Xenopus larvae are depolarized by GABA. To study the mechanisms underlying this distinctive response, intracellular and patch-clamp recordings were made from RB neurons in situ. The intracellularly recorded GABA reversal potential (EREV) was near -30 mV in normal saline and was approximately 25 mV more negative in Na(+)-free saline. Whole-cell recordings from RB neurons and from neighboring dorsolateral interneurons (DLi) revealed that GABA responses of both cells were mediated by GABAA receptors. Currents elicited by GABA were mimicked by muscimol and reversibly blocked by bicuculline, and EREV shifted with changes in Cl- concentration ([Cl]) in agreement with Cl- selectivity. In perforated patch recordings, EREV for RB cells was significantly more positive than for DLi cells (-38 vs -63 mV), indicating that intact RB cells maintain higher levels of intracellular Cl-. Replacement of external Na+ or exposure to the Cl- transport inhibitor bumetanide (100 microM) shifted RB cell EREV to move negative values, consistent with Na+(-)dependent Cl cotransport contributing to higher internal [Cl]. In contrast, these treatments did not change DLi cell EREV. The results indicate that a Na+(-)dependent Cl- transport mechanism underlies GABAA receptor-mediated depolarizing Cl- conductances in RB neurons. Thus, both inhibitory and excitatory GABA responses appear to be present during the same developmental period in vivo. GABA may stimulate Ca2+ influx in RB neurons because the intracellular GABA EREV is above the threshold for low voltage- activated Ca2+ channels.