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 superfamily of secondary active uptake systems for neurotransmitters that depend on the electrochemical gradients for Na+ and Cl-. In the GAT1, two Na+ ions and one Cl- ion are co-transported with one GABA molecule. Steady-state transport activity and transient charge movements during partial reactions of the 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 [3H]GABA uptake. Effects of mutation of two out of three N-glycosylation sites located in the extracellular loop between transmembrane domains 3 and 4 (Asn176, Asn181, Asn184) were analysed. Simultaneous substitution of two Asn by Asp leaves the transport system intact but leads to a reduction in turnover and complex changes in the interaction of external Na+ with the transport protein. If Asn176 is mutated to Asp and simultaneously Asn181 to Gly, no transport and no charge movements can be detected. In conclusion, mutations of the glycosylation sites result in altered transport, and the local conformation at Asn181 is critical for expression of transport activity.
Liu
Y
Y
Max-Planck Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt/Main, Germany.
Eckstein-Ludwig
U
U
Fei
J
J
Schwarz
W
W
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D006031:Glycosylation; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D016296:Mutagenesis; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D027361:Organic Anion Transporters; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
D002352:Carrier Proteins; D017931:DNA Primers; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493414:Slc6a1 protein, mouse; D012964:Sodium