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

Abstract

The H+-K+-ATPase α-subunit (HKα2) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα2plasma 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 Gs-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 HKα2expression 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 CO2. PKA activity and HKα2protein were monitored by immunoblot analysis, and HKα2mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα2mRNA; however, PKA activity and HKα2protein 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 HKα2expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα2protein by increasing PKA activity without altering HKα2mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα2and acid-base balance.