Effect of chemical thio modification internal cysteines of the SKOR channel

CARLOS GARCIA-MATA; PAWEL GAJDANOWICZ; INGO DREYER; MICHAEL R. BLATT

Manchester, UK

Congreso; SEB Plant Transport Group Meeting 2008; 2008

Plant ouward-rectifying K+ channels mediate K+ efflux from guar cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K+). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the c urrent are derived froom inside the cell. This K+ dependence confers a sensitivity to the extracellular concentration ([K+]) that ensuresthat the cahnnels mediate K+ efflux ony, regardless of the [K+] prevailing outside. With the aim of understanding the mechanisms of gating and its integration with voltage, we carried out two complementary strategies to asses the importance of the different cysteine residues on V and k+ dependent SKOR gating. The SKOR channel has 11 naturally existent cysteines, 3 o fthem are located within the membrande spans, one inthe S3 domain (C1) and 2 in the S5 domain (C2,3), the rest of them are located along the C-terminal tail inthe cytoplasm. We transfected HEK 293 cells with wild type channels and the mutants of the naturally oexistent cysteines, and we analyzed by patch clamp under whole cell configuration. Substituting only C1 or C2,3 gave current that resemble those of the Wt channel. In order to asses for those cysteines which hs importance fo voltage gating we created substitutional however, they don{t show the same response upon chemical thiol modification by using MTS reagents. The membrane permeant methyl methanethiosulfonate (MMTS), but ot the membrane impermeant aminoethyl methanethiosufonate, hydrobromide (MTSEA) or sodium (2-sufonatoethyl) methanethisulfonate (MTSES) reagent, poroduced a fast inactivation of voltage gated currens in Wt anmd C1 but not in the C2,3 mutants. This data agreed with preliminary data showing that SKOR channel is very sensible to mutation inthe Nt and central region of the S5 domian, and shows that C2 and/or C3 are key residues for V dependent gaiting