Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: characterization of Kat1, an inward-rectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast channels and carriers.
Transport-deficient strains of the yeast Saccharomyces cerevisiae have recently proven useful for cloning, by functional complementation, of cDNAs encoding heterologous membrane transporters: specifically, H(+)-amino acid symporters and K+ channels from the higher plant Arabidopsis thaliana. The present study uses whole-cell patch-clamp experiments to show that yeast strains which grow poorly on submillimolar K+ due to the deletion of two K(+)-transporter genes (TRK1 and TRK2) are in fact missing a prominent K+ inward current present in wild-type cells. Rescue of such strains for growth on low K+ by transformation with a gene (KAT1) encoding an inward-rectifying K+ channel from Arabidopsis is accompanied by the appearance of an inward current whose characteristics are in qualitative agreement with previous studies in the Xenopus oocyte system, but differ in quantitative details. The ability to make such measurements directly on Saccharomyces should facilitate structure-function studies of any electrogenic or electrophoretic ion transporters which can be expressed in the plasma membrane (or tonoplast) of that organism.
Bertl
A
A
Department of Cellular and Molecular Physiology, Yale University, New Haven, CT 06510, USA.
Anderson
J A
JA
Slayman
C L
CL
Gaber
R F
RF
D000818:Animals; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins; D002462:Cell Membrane / Q000502:physiology; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D018076:DNA, Complementary; D005260:Female; D005800:Genes, Fungal; D017343:Genes, Plant; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010940:Plant Proteins; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D012964:Sodium / Q000494:pharmacology; D014982:Xenopus laevis
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; C501210:KAT1 protein, Arabidopsis; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012964:Sodium; D011188:Potassium