Basolateral Na+/HCO3- cotransport activity is regulated by the dissociableNa+/H+ exchanger regulatory factor

In the renal proximal tubule, the activities of the basolateral Na+/HCO3- c otransporter (NBC) and the apical Na+/H+ exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated in hibition of NHE3 is mediated by a PDZ motif-containing protein, the Na+/Hexchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determin e whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cor tex immunoblot analysis using anti-peptide antibodies directed against rabb it NHE-RF demonstrated the presence of this regulatory factor in both brush -border membranes (BBMs) and basolateral membranes (BLMs). Using a reconsti tution assay, we found that limited trypsin digestion of detergent solubili zed rabbit renal BLM preparations resulted in NBC activity that was unaffec ted by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restore d the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE- RF also failed to attenuate NBC activity. To establish further a physiologi c role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-I, which lacks detectable endogenous NHE-RF expression, was engineered to exp ress stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) e xhibited markedly lower basal levels of NBC activity than did wild-type con trols. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 mu M of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only su ggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activiti es may be coordinately regulated in renal proximal tubule cells.