Poster: Late and Moved Abstracts

Abs # 962: Gating and permeation of the Saccharomyces K⁺ channel, Tok1p, are controlled by distinct amino acids within the two pore loops

Presenter:	Bertl, Adam , adam.bertl@chem-bio.uni-karlsruhe.de
Authors	Roller, Anja  (A)   Natura, Gabriel  (B)   Bihler, Hermann  (A)   Clifford, Slayman L. (C)   Bertl, Adam  (A)
Affiliations:	(A): Botany I, University of Karlsruhe (B): Physiology I, University of Jena (C): Medical School, Yale University

Gating of the outwardly rectifying 'two-pore' K⁺ channel, Tok1p, in the yeast plasma membrane is sensitive to membrane voltage and extracellular K⁺ concentration ([K⁺]_o), so that changes of [K⁺]_o produce nearly Nernstian shifts of the steady-state current-voltage characteristics. In high K⁺ (150 mM), channel activation is slow and shows only a slight trend of inactivation during sustained membrane depolarisation. Activation is faster and inactivation conspicuously large following reduction of [K⁺]_o to 15 mM. In order to analyse the mechanisms underlying K⁺-dependent activation and inactivation (or deactivation), we constructed channel mutants bearing single amino acid substitutions within the conserved pore loops, and expressed those mutants in a null strain for tok1, which was also deleted of the main K⁺ uptake carriers, Trk1p and Trk2p. The transformed yeast cells were analysed for K⁺-dependent growth and for altered gating and permeation of potassium via the expressed mutant Tok1 channel. We could thus identify several amino acid residues which are essential for cation sensing/binding and for stability of the conducting pathway.