Mutational analysis of basic residues in the rat vesicular acetylcholine transporter - Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation

The function of positively charged residues and the interaction of positive ly and negatively charged residues of the rat vesicular acetylcholine trans porter (rVAChT) were studied. Changing Lys-131 in transmembrane domain heli x 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on ve samicol binding. However, replacement by His or Arg retained transport acti vity, suggesting a positive charge in this position is critical. Mutation o f His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop h istidines showed no change in acetylcholine transport. The mutant H338A/D39 8N between oppositely charged residues in transmembrane domains showed no v esamicol binding, however the charge reversal mutant H338DID398H restored b inding, This suggests that His-338 forms an ion pair with Asp-398. The char ge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D42 5K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.