Developmental expression of ROMK in rat kidney

The apical secretory K⁺ (SK) channel in the principal cell represents the rate-limiting step for K⁺ secretion across the cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit principal cells identifies an increase in number of conducting SK channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol. 272 (Renal Physiol. 41): F397- F404, 1997], ~1 wk after an increase in activity of the amiloride-sensitive epithelial Na⁺ channel (ENaC) is detected. To correlate the postnatal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to show a developmental increase in abundance of renal ROMK mRNA and a ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney. Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of the neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody directed against distal and connecting tubule (CNT)-specific kallikrein or the lectin Dolichos biflorus agglutinin (DBA), which labels proximal tubules and collecting ducts. In inner cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA- positive collecting ducts. By 3 wk of age, coincident ROMK and DBA expression was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revealed a developmental increase in a ~40-kDa band, the expected size for ROMK. Immunolocalization of α-ENaC showed apical staining of a majority of cells in distal nephron segments after the 1st week of postnatal life. The βand γ-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance; γ-ENaC showed some apical polarization. These results suggest that the postnatal increases in a principal cell apical SK and Na⁺ channel activity are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins.