Molecular cloning and expression characterization of a rice K+ channel β subunit

K⁺ channel proteins native to animal membranes have been shown to be composed of two different types of polypeptides: the pore-forming subunit and the subunit which may be involved in either modulation of conductance through the channel, or stabilization and surface expression of the channel complex. Several cDNAs encoding animal K⁺ channel subunits have been recently cloned and sequenced. We report the molecular cloning of a rice plant homolog of these animal subunits. The rice cDNA (KOB1) described in this report encodes a 36 kDa polypeptide which shares 45% sequence identity with these animal K⁺ channel subunits, and 72% identity with the only other cloned plant (Arabidopsis thaliana) K⁺ channel subunit (KAB1). The KOB1 translation product was demonstrated to form a tight physical association with a plant K⁺ channel subunit. These results are consistent with the conclusion that the KOB1 cDNA encodes a K⁺ channel subunit.Expression studies indicated that KOB1 protein is more abundant in leaves than in either reproductive structures or roots. Later-developing leaves on a rice plant were found to contain increasing levels of the protein with the flag leaf having the highest titer of KOB1. Leaf sheaths are known to accumulate excess K⁺ and act as reserve sources of this cation when new growth requires remobilization of K⁺. Leaf sheaths were found to contain higher levels of KOB1 protein than the blade portions of leaves. It was further determined that when K⁺ was lost from older leaves of plants grown on K⁺-deficient fertilizer, the loss of cellular K⁺ was associated with a decline in both KOB1 mRNA and protein. This finding represents the first demonstration (in either plants or animals) that changes in cellular K⁺ status may specifically alter expression of a gene encoding a K⁺ channel subunit.     

Guanylyl cyclase stimulatory coupling to K(Ca) channels

We coexpressed the human large-conductance, calcium-activated K(K_Ca) channel (- and -subunits) and rat atrialnatriuretic peptide (ANP) receptor genes in Xenopus oocytesto examine the mechanism of guanylyl cyclase stimulatory coupling tothe channel. Exposure of oocytes to ANP stimulated whole cellK_Ca currents by 21 ± 3% (at 60 mV), without alteringcurrent kinetics. Similarly, spermine NONOate, a nitric oxide donor,increased K_Ca currents (20 ± 4% at 60 mV) in oocytesexpressing the channel subunits alone. Stimulation of K_Cacurrents by ANP was inhibited in a concentration-dependent manner by apeptide inhibitor of cGMP-dependent protein kinase (PKG).Receptor/channel stimulatory coupling was not completely abolished bymutating the cAMP-dependent protein kinase phosphorylation site on the-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI.J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residuesvirtually abolished coupling. Spermine NONOate also failed to stimulatechannels expressed from the double mutant cRNAs. These data indicatethat nitric oxide donors stimulate K_Ca channels throughcGMP-dependent phosphorylation and that two serine residues (855 and869) underlie this stimulatory coupling.