Juan Codina

Expression of HKα2 protein is increased selectively in renal medulla by chronic hypokalemia

Our laboratory has demonstrated by Northern analysis that chronic hypokalemia increases HKα2 (i.e., α-subunit of the colonic H+-K+-ATPase) mRNA abundance in the rat. To determine whether the increase in mRNA correlated with an increase in HKα2 protein, an antibody was raised against a synthetic peptide derived from amino acids 686-698 of the HKα2sequence. The anti-HKα2 antibody hybridized to rat distal colon membranes which migrated at ∼100 kDa (expected mobility of HKα2). HKα2 protein was not detected in plasma membranes from rat whole kidney or stomach (100 μg) derived from control animals. The antibody was then used to investigate changes in expression of HKα2 in renal cortex, renal medulla, and distal colon in two pathophysiological conditions: 1) chronic hypokalemia (LK) and 2) chronic metabolic acidosis (CMA). In LK rats there was a marked, but selective, increase in the abundance of HKα2 protein in membranes prepared from renal medulla. Nevertheless, a corresponding increase in HKα2 protein abundance was not observed in membranes prepared from the distal colon of LK rats. HKα2 protein abundance in CMA was indistinguishable from controls. Moreover, chronic hypokalemia had no effect on expression of α1-Na+-K+-ATPase or HKα1 in kidney or distal colon under any experimental condition. Therefore, HKα2 protein is tissue- and site-specifically upregulated in response to chronic hypokalemia but not by CMA. Furthermore, this regulatory response is localized to the renal medulla.

H+,K+-ATPase.

At least four unique H,K-ATPases have been cloned and are expressed in several tissues. Recent findings have enhanced our appreciation of the roles of H,K-ATPases in the kidney with respect to their molecular identities, functional properties, segmental and intrarenal distribution, and regulatory features. The major role of the H,K-ATPases expressed in mammalian kidney, the gastric and colonic H,K-ATPases, is to regulate potassium and bicarbonate absorption in collecting duct segments.

Phosphorylation of S955 at the Protein Kinase A Consensus Promotes Maturation of the α Subunit of the Colonic H+,K+-ATPase

All the alpha subunits of the Na+,K+ -ATPases and H+,K+ -ATPases have a protein kinase A (PKA) consensus sequence near or in the ninth transmembrane domain. The role of this domain in influencing alpha subunit synthesis/degradation, plasma membrane localization, and 86Rb+ uptake has not been established for the alpha subunit of the colonic H+,K+ -ATPase. This study examined the effect of mutating S955 (within the PKA consensus site of the alpha subunit of the colonic H+,K+ -ATPase [HKalpha2]) to alanine (S955/A) or aspartic acid (S955/D) on alpha subunit expression and function. The results demonstrate that a negatively charged amino acid at position 955 of HKalpha2 promotes higher expression levels of both whole-cell and plasma membrane-localized HKalpha2. Moreover, inhibition of PKA reduced expression of wild-type HKalpha2 and associated 86Rb+ uptake. Last, the activity of the HKalpha2 S955/A was rescued by treatment with 4-phenylbutyric acid, a compound that was shown previously to restore function to the cystic fibrosis transmembrane conductance regulator.

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2)

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.