Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter

The GABA (gamma-aminobutyric acid) transporter (GAT1) belongs to a superfam ily of secondary active uptake systems for neurotransmitters that depend on the electrochemical gradients for Na+ and Cl-. In the GAT1, two Na+ ions a nd one Cl- ion are co-transported with one GABA molecule. Steady-state tran sport activity and transient charge movements during partial reactions of t he transport cycle of the GAT1 of mouse brain expressed in Xenopus oocytes were investigated by two-electrode voltage clamp. Functional expression was demonstrated by Na+-dependent [H-3]GABA uptake. Effects of mutation of two out of three N-glycosylation sites located in the extracellular loop betwe en transmembrane domains 3 and 4 (Asn(176), Asn(181), Asn(184)) were analys ed. Simultaneous substitution of two Asn by Asp leaves the transport system intact but leads to a reduction in turnover and complex changes in the int eraction of external Na+ with the transport protein. If Asn(176) is mutated to Asp and simultaneously Asn(181) to Gly, no transport and no charge move ments can be detected, In conclusion, mutations of the glycosylation sites result in altered transport, and the local conformation at Asn(181) is crit ical for expression of transport activity. (C) 1998 Elsevier Science B.V. A ll rights reserved.