Ectopic GABA_A receptor β3 subunit determines Cl^- / -ATPase and chloride transport in HEK 293FT cells

Neuronal intracellular chloride concentration ([Cl^- ]_i ) is a crucial determinant of transmission mediated by the γ-aminobutyric acid type A receptor (GABA_A R), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl^- ion is the main carrier of charge through the GABA_A R; however, bicarbonate ions ( $$ ) flowing in the opposite direction can also contribute to the net current. The direction of Cl^- and $$ fluxes is determined by the underlying electrochemical gradient, which is controlled by Cl^- transporters and channels. Accumulating evidence suggests that active mechanisms of chloride transport across the GABA_A R pore can underlie the regulation of [Cl^- ]_i . Measurement of Cl^- / $$ -ATPase activity and Cl^- transport in HEK 293FT cells expressing homomeric or heteromeric GABA_A R ensembles (α2, β3, or γ2) with fluorescent dye for chloride demonstrated that receptor subtypes containing the β3 subunit show enzymatic activity and participate in GABA-mediated or ATP-dependent Cl^- transport. GABA-mediated flow of Cl^- ions into and out of the cells occurred for a short time period but then rapidly declined. However, Cl^- ion flux was stabilized for a long time period in the presence of $$ ions. The reconstituted β3 subunit isoform, purified as a fusion protein, confirmed that β3 is critical for ATPase; however, only the triplet variant showed the full receptor function. The high sensitivity of the enzyme to γ-phosphate inhibitors led us to postulate that the β3 subunit is catalytic. Our discovery of a GABA_A R type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.