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The inwardly rectifying potassium channel Kir 2.3 is specifically targeted and expressed on the basolateral membrane of certain renal epithelial cells. In the present study, the structural basis for polarized targeting was elucidated. Deletion of a unique COOH-terminal domain produced channels that were mistargeted to the apical membrane, consistent with the removal of a basolateral membrane-sorting signal. By characterizing a series of progressively smaller truncation mutants, an essential targeting signal was defined (residues 431-442) within a domain that juxtaposes or overlaps with a type I PDZ binding motif (442-445). Fusion of the COOH-terminal structure onto CD4 was sufficient to change a random membrane-trafficking and expression pattern into a basolateral membrane one. Using metabolic labeling and pulsechase and surface immunoprecipitation, we found that CD4-Kir2.3 COOH-terminal chimeras were rapidly and directly targeted to the basolateral membrane, consistent with a sorting signal that is processed in the biosynthetic pathway. Collectively, the data indicate that the basolateral sorting determinant in Kir 2.3 is composed of a unique arrangement of trafficking motifs, containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based signals. The previously unrecognized domain corresponds to a highly degenerate structure within the Kir channel family, raising the possibility that the extreme COOH terminus of Kir channels may differentially coordinate membrane targeting of different channel isoforms.

Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK. Am J Physiol Cell Physiol 280: C192-C198, 2001.--[Na.sup.+]/[H.sup.+] exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on [Na.sup.+]/[H.sup.+] exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney. sodium hydrogen exchanger regulatory protein 2; sodium hydrogen exchanger 3 protein kinase A regulatory protein; signal complexes; PDZ proteins

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging. Am J Physiol Renal Physiol 319: F754-F764, 2020. First published September 14, 2020; doi:10.1152/ajprenal.00441.2020.-- The thiazide-sensitive Na+-Cl cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, stateof- the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics. [ABSTRACT FROM AUTHOR]

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a Gq protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl−/HCO3− exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl− absorption, we examined α-KG-stimulated Cl− absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl− absorption in CCDs from wild-type mice but had no effect on Cl− absorption in pendrin knockout mice. Because Gq protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl− absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl− absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl− absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca2+ chelators (BAPTA, 10–20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl− absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl− absorption. However, in SPAK null CCDs, Cl− absorption was not activated further by luminal α-KG application nor was Cl− absorption reduced with the PKC inhibitor GF109203. Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl−/HCO3− exchange through a mechanism dependent on PKC and Ca2+ that involves PKC-α and PKC-δ. [ABSTRACT FROM AUTHOR]

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+- ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2. [ABSTRACT FROM AUTHOR]