ION CHANNELS Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K + channel, Tok1p Anja Roller & Gabriel Natura & Hermann Bihler & Clifford L. Slayman & Adam Bertl Received: 24 September 2007 / Revised: 9 December 2007 / Accepted: 2 January 2008 # Springer-Verlag 2008 Abstract Tandem pore-loop potassium channels differ from the majority of K + channels in that a single polypeptide chain carries two K + -specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP 1 M–MP 2 M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P 1 and Y424 in P 2 ) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal con- ductance when expressed in Saccharomyces itself, but the P 1 mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P 1 than at P 2 appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P 2 (overwhelmingly TIGYGD) to be much stabler than that of P 1 (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)—which inverts the order of residues from the wild-type channel —reduces the expressed channel conductance by a large factor (20-fold, cf. <twofold for the single mutants). Keywords Voltage dependence . Conductance . Potassium channel . Whole-cell recording . Activation kinetics Introduction The majority of potassium-selective ion channels are thought to be assembled symmetrically around a homotet- rad of α subunits. Each of these possesses a core structure of two transmembrane α helices (TM segments, or just M segments) sandwiching a recurrent loop which bears the K + -channel filter sequence: TVGYGD in most voltage- dependent channels and in the crystallized bacterial K + channel KcsA [9]. The functional potassium filter, includ- ing up to four K + -binding sites (labeled S 1 ,S 2 ,S 3 ,S 4 , inward from the membrane outer surface) is formed by the symmetric apposition of such filter sequences within each tetrad assembly. The recurrent loop, generally referred to as a P loop (pore loop, or pore motif), serves the additional Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-008-0446-0 A. Roller : H. Bihler : A. Bertl Botanisches Institut I, Universität Karlsruhe (TH), Kaiserstrasse 12, 76128 Karlsruhe, Germany G. Natura Physiologisches Institut, Universität Jena, Teichgraben 8, 07740 Jena, Germany C. L. Slayman Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA Present address: A. Bertl (*) Botanisches Institut, Fachbereich Biologie, TU Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany e-mail: bertl@bio.tu-darmstadt.de