RENAL CORTICAL BASOLATERAL NA+ HCO3- COTRANSPORTER .3. EVIDENCE FOR AREGULATORY PROTEIN IN THE INHIBITORY EFFECT OF PROTEIN-KINASE-A/

The activity of the Na-H antiporter is inhibited by cyclic AMP-depende nt protein kinase A (cAMP-PKA). The inhibitory effect of PKA on the Na -H antiporter is mediated through a regulatory protein that can be dis sociated from the antiporter by limited protein digestion. PKA also in hibits the activity of the Na+/ HCO3- cotransporter. We investigated w hether the activity of Na+/HCO3- cotransporter and the effect of PKA o n this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubiliz ed BLM reconstituted in liposomes (proteoliposomes), trypsin (100 mu g ) increased Na-22 uptake in the presence of HCO3 but not in the presen ce of gluconate, indicating that trypsin does not alter diffusive Na-2 2 uptake but directly stimulates the Na+/HCO3- cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CS U) of cAMP-PKA decreased the activity of the Na+/HCO3- cotransporter ( expressed as nanomoles/mg protein/3s) from 23 +/- 10 to 14 +/- 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAM P-PKA on the activity of Na+/HCO3- cotransporter was blunted. To ident ify a fraction that was responsible for the inhibitory effect of the C SU on the Na+/HCO3- cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP- PKA on the Na+/CO3- cotransporter activity was assayed in proteoliposo mes digested with trypsin with the addition of a fraction containing t he 42 kDa protein (fraction S+) or without the 42 kDa protein (fractio n S-). With the addition of fraction S-, the CSU of cAMP-PKA failed to inhibit the Na+/HCO3- cotransporter activity (control 27 +/- 6, CSU 2 7 +/- 3) while the addition of fraction S+ restored the inhibitory eff ect of CSU (27 +/- 6 to 3 +/- 0.3 P < 0.01). The CSU of cAMP-PKA phosp horylated several proteins in solubilized protein including a 42 kDa p rotein. Fluorescein isothiocyanate (FITC) labels components of the Na/HCO3- cotransporter including the 56 kDa and 42 kDa proteins. In tryp sin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the N a+/HCO3- cotransporter is regulated by protein(s) which mediates the i nhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.