Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells.
A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than -60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+in currents half-maximally with an inhibitor concentration (IC50) of 105 microM. An almost identical high Cs+ sensitivity (IC50 = 90 microM) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.
Zimmermann
S
S
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, Haus 20, D-14476 Golm/Potsdam, Germany.
Talke
I
I
Ehrhardt
T
T
Nast
G
G
Müller-Röber
B
B
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015415:Biomarkers; D002460:Cell Line; D002586:Cesium / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004817:Epidermis / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000032:analysis; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D018517:Plant Roots / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D024661:Potassium Channels, Inwardly Rectifying; D011198:Solanum tuberosum / Q000235:genetics*; D018411:Spodoptera / Q000737:chemistry / Q000166:cytology / Q000235:genetics*
D029681:Arabidopsis Proteins; D015415:Biomarkers; D018076:DNA, Complementary; C501210:KAT1 protein, Arabidopsis; D008164:Luminescent Proteins; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D049452:Green Fluorescent Proteins; D002586:Cesium