Guanylyl cyclase stimulatory coupling to K-Ca channels

We coexpressed the human large-conductance, calcium-activated K (K-Ca) chan nel (alpha- and beta -subunits) and rat atrial natriuretic peptide (ANP) re ceptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclas e stimulatory coupling to the channel. Exposure of oocytes to ANP stimulate d whole cell K-Ca currents by 21 +/- 3% (at 60 mV), without altering curren t kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K- Ca currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K-Ca currents by ANP was inhibited in a concentratio n-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished b y mutating the cAMP-dependent protein kinase phosphorylation site on the al pha -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 si te (S855), but mutation of both residues virtually abolished coupling. Sper mine NONOate also failed to stimulate channels expressed from the double mu tant cRNAs. These data indicate that nitric oxide donors stimulate K-Ca cha nnels through cGMP-dependent phosphorylation and that two serine residues ( 855 and 869) underlie this stimulatory coupling.