Ectopic GABAA receptor β3 subunit determines Cl−/HCO3-‐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 (GABAAR), which subserves synaptic and extrasynaptic inhibition as well as excitation. The Cl− ion is the main carrier of charge through the GABAAR; 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 GABAAR 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 GABAAR 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 GABAAR type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling. [ABSTRACT FROM AUTHOR]