Ectopic GABA A receptor β3 subunit determines Cl ‐ /HCO 3 ‐ ‐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 (GABAA R), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl- ion is the main carrier of charge through the GABAA R; however, bicarbonate ions (HCO3- ) flowing in the opposite direction can also contribute to the net current. The direction of Cl- and HCO3- 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 GABAA R pore can underlie the regulation of [Cl- ]i . Measurement of Cl- /HCO3- -ATPase activity and Cl- transport in HEK 293FT cells expressing homomeric or heteromeric GABAA 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 HCO3- 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 GABAA R type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.