Electrophysiology of Ammonium Transport Proteins

Abstract

The transport of ammonium/ammonia across biological membranes is mediated by a family of ubiquitous integral membrane proteins, the Ammonium Transport proteins (Amt) [1]. Current functional interpretations derived from electrophysiology (two-electrode voltage clamp experiments with protein expressed in Xenopus oocytes [2,3]), in vivo radioactive uptake assays (using methylammonium as alternative substrate [4]) and in vitro pH measurements (reported by a pH-sensitive Fluorescence dye inside proteoliposomes [5]), yielded to discrepant results that are still controversially discussed [1,6,7]. To conclude on the substrate identity (NH4+ or NH3) and the transport mechanism, we are conducting electrophysiology assays on various Amt proteins. Typically, pure protein is reconstituted in liposomes for Planar Lipid Bilayer (PLB) and Solid Supported Membrane (SSM) experiments. So far, assays in PLBs show protein fusion difficulties or weak signals even under macroscopic current recording conditions. On the contrary, SSMs allow testing for higher protein:lipid ratios in a much more stable setup [8]. Under these conditions we have been able to record transient currents upon protein activation with substrate. We are currently further developing this assay to investigate electrogenic ammonium uptake in Af-Amt1.[1] S. L. A. Andrade et al, Mol Membr Biol, 2007, 24, 357.[2] M. Maier et al, Biochem J, 2006, 396, 431.[3] C. Ortiz-Ramirez et al, J Biol Chem, 2011, 286, 31113.[4] E. Soupene et al, PNAS, 1998, 95, 7030.[5] S. Khademi et al, Science, 2004, 305, 1587.[6] A. Javelle et al, J Struct Biol,2007, 158, 472.[7] R. N. Fong et al, PNAS,2007, 104, 18706.[8] P. Schulz et al., Methods, 2008, 46, 97.