Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demonstrated 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 al., 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 functional similarities and functional differences: similarities in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replacement, in the affinities of blocking ions, and in dependence of gating kinetics on extracellular K(+). Kat1, previously described in yeast (Bertl et al., 1995), is about 20-fold more permeable to K(+) than to Na(+) or NH(+)(4), 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(+)(4), 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(+)(4) reduces subsequent K(+) currents by nearly 75%. Furthermore, lowering of extracellular K(+) concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinetics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Ypk1 (Duk1; Reid et al., 1996). The data suggest that all of the ions tested bind within the open channels, such that the weakly permeant species (Na(+), NH(+)(4)) 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.

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Journal Article

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Bertl A, Reid JD, Sentenac H, Slayman CL

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