Characterization of Na 1 / HCO 3 2 cotransporter isoform NBC - 3

cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by ,90% but increased the Na1dependent HCO3 2 cotransport activity by ,7-fold (as assayed by DIDS-sensitive, Na1-dependent, HCO3 -mediated intracellular pH recovery). This increase was associated with ,5.5fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li1 in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting HCO3 2 from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of HCO3 2 into the cells.