γ-hydroxybutyrate receptor function determined by stimulation of rubidium and calcium movements from NCB-20 neurons

Abstract

γ-Hydroxybutyrate is derived from GABA in brain and plays specific functional roles in the CNS. It is thought to exert a tonic inhibitory control on dopamine and GABA release in certain brain areas, through specific γ-hydroxybutyrate receptors. Apart from modifying certain calcium currents, the specific transduction mechanism induced by stimulation of γ-hydroxybutyrate receptors remains largely unknown. We investigated the possible contribution of K + channels to the hyperpolarization phenomena generally induced by γ-hydroxybutyrate in brain, by monitoring 86Rb + movements in a neuronal cell line (NCB-20 cells), which expresses γ-hydroxybutyrate receptors. Physiological concentrations of γ-hydroxybutyrate (5–25 μM) induce a slow efflux of 86Rb +, which peaks at 5–15 min and returns to baseline levels 20 min later after constant stimulation. This effect can be reproduced by the γ-hydroxybutyrate receptor agonist NCS-356 and blocked by the γ-hydroxybutyrate receptor antagonist 6,7,8,9-tetrahydro-5-[ H]-benzocycloheptene-5-ol-4-ylidene. The GABA B receptor antagonist CGP 55,845 has no effect on γ-hydroxybutyrate-induced 86Rb + efflux. The pharmacology of this γ-hydroxybutyrate-dependent efflux of 86Rb + is in favor of the involvement of tetraethylammonium and charybdotoxin insensitive, apamin sensitive Ca 2+ activated K + channels, identifying them as small conductance calcium activated channels. We demonstrated a γ-hydroxybutyrate dose-dependent entry of calcium ions into NCB-20 neuroblastoma cells at resting potential. Electrophysiological data showed that this Ca 2+ entry corresponded mainly to a left-hand shift of the current/voltage relation of the T-type calcium channel. This process must at least partially trigger small conductance calcium activated channel activation leading to γ-hydroxybutyrate-induced hyperpolarization.