Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells

摘要

A cDNA encoding a novel, inwardly rectifying K+ (K+in) channel protein, SKT1, was cloned from potatoes (Solanum tuberosum) cv. Desiree. SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potatoes. Skt1 mRNA was 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 105μM. An almost identical high Cs+ sensitivity (IC30 = 90μM) 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. Nucleotide sequence data have been submitted to the EMBL/GenBank/DDBJ databases under the accession number X86021.