Journal of Experimental Botany, Vol. 48, Special Issue, pp. 405-413, March 1997 Journal of Experimental Botany Functional comparison of plant inward-rectifier channels expressed in yeast Adam BertI 1 ,5 , John D. Reid 2 , Herve Sentenac 3 and Clifford L. Siayman 4 1 Botanisches Institut I, Universitet Karlsruhe, Kaiserstrasse 2, D-76128 Karlsruhe, Germany 2 G/axo Institute for Molecular Biology, CH-1228 Plan-Ies-Ouates, Geneva, Switzerland 3 Biochimie et Physiologie Vegetales, ENSA-M/INRA/CNRS, F-34060 Montpellier cedex 1, France 4 Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA Received 30 April 1996; Accepted 14 Octeber 1996 Abstract Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demon- strated by the successful screening of plant cDNA libraries for complementation of transport defects in especially constructed strains of yeast. The first experiments of this sort identified two potassium- channel genes from Arabidopsis thaliana, designated KAT1 and AKT1 (Anderson et al., 1992; Sentenac et et., 1992), both of which code for proteins resembling the Shaker superfamily of K+ channels in animal cells. Patch-clamp analysis, directly in yeast, of the two channel proteins (Kat1 and Akt1) reveals both func- tional similarities and functional differences: similarit- ies in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replace- ment, in the affinities of blocking ions, and in depend- ence of gating kinetics on extracellular K+• Kat1, previously described in yeast (Berti et et., 1995), is about 20-fold more permeable to K+than to Na+ or NH: , shows K+-independent gating kinetics, and is blocked with moderate effectiveness (30-50% at 10 mM) by barium and tetraethylammonium (TEA +) ions. Akt1, by contrast, is weakly inhibited by TEA +, more strongly inhibited by Ba 2 :- , and very strongly inhibited by Cs +. Furthermore Na + and NH: , while having about the same permeance to Akt1 as to Kat1, have delayed effects on Akt1: brief replacement of extracellular K+ by Na+ enhances by nearly 100% the subsequent K+ currents after sodium removal; and brief replacement of K+ by NH: reduces subsequent K+ currents by nearly 75%. Furthermore, lowering of 5 To whom correspondence should be addressed. Fax: +49 721 6084193. © Oxford, University Press 1997 extracellular K+ concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinet- ics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Vpk1 (Duk1; Reid et al., 1996). The data suggest that all of the ions tested bind within the open channels, such that the weakly per- meant species (Na +, NH:) are easily displaced by K+, but the blocking species (Cs +, Ba 2+, TEA +) are not easily displaced. With Akt1 , furthermore, the permeant ions bind to a modulator site where they persist after removal from the medium, and through which they can alter the channel conductance. Extracellular K+ itself also binds to a modulator site, thereby enhancing the rate of opening of Akt1. Key words: Plant ion channels, yeast, cDNA library, screen- ing, patch clamp analysis. Introduction Cloning of transporter genes in higher plants has revealed that apparently simple transport functions can involve many genetically distinct proteins. For example, in the small crucifer Arabidopsis thaliana, at least 10 genes code for variants of the plasma membrane proton pump (Harper et aI., 1994). The biological purpose of such apparent redundancy is unknown, although tissue specialization and developmental staging are plausible explanations. Studies of potassium channels in the same organism, A. thaliana, have revealed a similar pattern of genetic by guest on October 13, 2011 jxb.oxfordjournals.org Downloaded from