Identification and functional characterisation of a class on non-selective cation channels in Saccharomyces cerevisiae.

Abstract

Na⁺ accumulation is a significant component in salt toxicity in plants. Although many proteins that facilitate Na⁺ flux have been identified in plants, investigations aimed at identifying the initial mechanism of Na⁺ entry into plants have failed. The catalysis of rapid, high capacity flux of Na⁺ across plant cell membranes, currently through unknown means, is of particular importance. Observations of Na⁺ flux across cellular membranes, using techniques such as electrophysiology and radiotracer flux, suggests this is a protein-based catalysis (Demidchik, 2002a; Demidchik and Tester, 2002c) (Essah et al., 2003). Based on these data, a class of protein described as voltage insensitive non-selective cation channel(s) (viNSCCs) are considered a good candidate for this Na⁺ flux. This is due in part to characteristics they possess. These characteristics include catalysis of high capacity / low affinity cation flux, being relatively non-selective to the point where Na⁺, K⁺, NH₄⁺ and MA⁺ flux with similar properties and this flux being sensitive to elevated Ca²⁺ and changes in pH. A screen using the toxic ammonium analogue methylammonium (MA⁺) was developed using Saccharomyces cerevisiae strains that have minimal high affinity ammonium uptake capability. An in silico screen was developed and a number of candidate genes were identified as being possible viNSCCs. Preliminary selection of these was then conducted using the developed S. cerevisiae screen. Two genes, belonging to the same protein family, were selected based upon these results. Analysis of these proteins using radiotracer flux in S. cerevisiae and electrophysiological examination using Xenopus laevis oocytes revealed these proteins catalyse the non-selective flux of mono-valent cations following unsaturable kinetics, indicative of a low affinity transport system. Further analysis revealed this cation flux is sensitive to external Ca²⁺. These properties strongly indicate these proteins form voltage insensitive non-selective cation channels in their native system.