Electrophysiology of Amt Proteins in Planar Lipid Bilayers

Abstract

The transport of ammonium/ammonia across biological membranes is mediated by a family of ubiquitous integral membrane proteins, the ammonium transport (Amt) proteins. Although high-resolution crystal structures of four Amt proteins are currently available [1-5], the substrate identity and transport mechanism are still controversially discussed [6].Current functional data using two-electrode voltage clamp experiments of protein expressed in Xenopus oocytes [7,8], measuring the radioactive uptake of methyl ammonium (an alternative substrate) in whole cells [9] or following pH variation with fluorescent probes in proteoliposomes [1], yielded to variable and discrepant results. To conclude on the substrate identity (NH4+ or NH3) and the transport mechanism we are carrying out electrophysiology measurements with reconstituted, pure protein in planar lipid bilayers (PLB). Such experiments have been crucial in identifying transport details in many proteins, among which, the elegant elucidation of Cl-/H+ antiport in CLC chloride channels [10] or, more recently, the pH gating mechanism in formate channels [11].If Amt proteins are electrogenic transporters/channels, charged substrate can be identified and followed by means of electric currents in PLB.[1] S. Khademi et al, Science, 2004, 305, 1587[2] L. Zheng et al, PNAS, 2004, 101, 17090[3] S. L. A. Andrade et al, PNAS, 2005, 102, 14994.[4] D. Lupo et al, PNAS, 2007, 104, 19303; X. Li et al, PNAS 2007, 104, 19279.[5] F. Gruswitz et al, PNAS, 2010, 107, 9638.[6] S.L.A. Andrade et al, Mol. Memb. Biol., 2007, 24, 357.[7] M. Maier M et al.,Biochem J., 2006, 396, 431.[8] C. Ortiz-Ramirez et al.,J Biol Chem., 2011, 286, 31113.[9] E. Soupene et al., PNAS, 1998, 95, 7030[10] A. Accardi et al., Nature. 2004, 427,803.[11] W. Lu et al., Science, 2011, 332, 352.