Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+channels

Abstract

Our objective was to identify and localize a K+channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to β-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at ∼50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+-K+-ATPase and ClC-2 Cl-channels. Function of native K+channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+channels exhibited a linear current-voltage relationship and a single-channel slope conductance of ∼11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Poto ∼0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+= Rb+>> Na+= Cs+= Li+= NMDG+. These findings strongly suggest that the Kir2.1 K+channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.