Five rice cDNAs that are most likely to encode a putative voltage-gated shaker type K+ channel were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of a well-preserved K+ selectivity filter (TXXTXGYG) in reference to Arabidopsis shaker type K+ channel (AKT1). The five candidate cDNAs were further subcloned into pSP64T vector, a Xenopus expression vector, and then in vitro transcribed to generate cRNAs. Each cRNAs were microinjected into Xenopus oocytes, and their K+ channel conductance was measured electrophysiologically by using two electrode voltage clamping (TEVC). Among them, one of the rice cDNAs gave rise to a K+ current with biophysical characteristics similar to those of the shaker type K+ channel.
Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir 6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel.
Collectively, cloning and functional characterization of rice and bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.
