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.

Abstract

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 (TRKI 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 (KATI) 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 structurefunction studies of any electrogenic or electrophoretic ion transporters which can be expressed in the plasma membrane (or tonoplast) of that organism.Over the past 10 years applied cellular genetics and molecular biology have revolutionized the study of biological membranes. The concepts of channel, carrier, and receptororiginally deduced from physical, chemical, and physiological measurements-have metamorphosed into pictures of families of protein molecules with defined primary structure. And a central topic of membrane research has become the functional analysis of mutant proteins whose structures are altered by systematic amino acid changes. Such structure-function studies lean heavily on techniques for expressing the proteins of interest in foreign membranes, where altered function can be examined more easily than in the native membranes (1-3).For proteins which convey ionic currents through membranes-i.e., ion channels and most ion pumps or ionsubstrate symporters-a prime tool of functional analysis has become the patch clamp, and the most widely adopted expression system has been mature oocytes of the African clawed frog Xenopus laevis, whose large size and easy handling have generally outweighed their prime disadvantage, that only transient expression is obtained.On the other hand, an attractive stable expression system which is excellent for primary genetic manipulation is the yeast Saccharomyces cerevisiae. Until recently, however, the small size of Saccharomyces cells and the peculiar mechanical properties of their membranes have made their electrophysiological study trying, despite the early demonstration (4) of the feasibility of patch-clamping yeast. Routine methods for recording from isolated patches of yeast membrane had also been described (5-7), but repeated attempts to identify heterologously expressed transporters in such patches had failed (A.B., unpublished work). This failure led us to extend the patchclamp technique with Saccharomyces to a stable whole-cell recording geometry, whose usefulness when applied in combination...

Functional expression of the plant K+ channel KAT1 in insect cells

“…The major difference of the in vitro studies of Schachtman et al [21] and Hedrich et al [12] to those reported here for Sf9 cells concerns the relative permeability of KAT1 for NH~ over K +. In contrast to the situation in oocytes but in line with results obtained in yeast [4], currents carried by NH~ were not significant in Sf9 cells. The lack of NH~-transport through KAT1 expressed in yeast has been erroneously related to block of the plant K + channel following NH] induced cytoplasmic acidification [29].…”

“…With respect to the expression system the threshold potential of KAT1 activation shifted along the voltage axis. Compared to the KAT1 properties in oocytes, the activation potential of KAT1 is less negative in Sf9 cells but more negative in yeast [4,12,25,26]. Furthermore, the shift of the activation potential in insect cells is accompanied by a shift of the deactivation kinetics towards more positive potentials.…”

“…In order to examine their electrical properties, expression systems were selected which tolerate transient membrane polarization in the range of-80 mV to -250 mV, characteristic for plant cells rather than for animal cells (-40 mV to -80 mV). So far, KAT1 could be functionally expressed in Xenopus oocytes and yeast [4,12,21,25,26], systems which allow the voltage-dependent stimulation of KAT1.…”

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

“…:fince the functional expression of the first plant sugar transporter in S. pombe [25] yeast cells became increasingly important for the expression cloning of many other carrier proteins [261 and recently also for the patch-clamp analysis of recombina~t channel proteins [27]. Large scale purification of proteins from yeast, however, was assumed to be limited in this organism [26].…”

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

“…For example, the K ϩ uptake defect of the yeast trk1trk2 deletion mutant prevents growth in K ϩ -limited medium. Functional complementation of this growth limitation has been successfully exploited for expression cloning, mutagenesis, and traf- ficking studies of a variety of unrelated K ϩ channels from plants and animals (2,6,79).…”