Electrophysiological Investigation of the Na+/H+ Antiporter NHAA from E. Coli.

Abstract

NhaA from E. coli serves as a prototype for Na+/H+ antiporters. It was proposed that the pH dependent change of the transport activity is due to the transition of the transporter from an inactive to an active conformation, which is allosterically controlled by a pH sensor.NhaA is electrogenic. Therefore it is possible to perform electrophysiological measurements with NhaA using the solid supported membrane based electrophysiology (Mager et al., 2011). The SSM based electrophysiology permits measurements with a high time resolution and defined conditions on both sides of the membrane.The kinetic analysis of the SSM measurements shows that the decrease of the transport activity at suboptimal pH values is due to the competitive binding of the substrates to a common binding site (Mager, et al., 2011). Based on this finding we employed a minimal kinetic model. It explains the substrate dependence of the transport activity over a large concentration range.The changed pH dependencies of the mutants V254C NhaA and H225R NhaA were explained with the impairment of the allosteric mechanism of inactivation (Padan, 2008). However the phenomenological alterations of the transport properties can be explained by our minimal kinetic model, which does not include an allosteric mechanism of pH regulation. The altered transport properties are the result of a change of the substrate binding affinities or the rates of the conformational transitions.1. Padan, E., Trends Biochem Sci 33, 435-443 (2008).2. Mager, T. et al., J Biol Chem 286, 23570-23581 (2011).