Localization of the ROMK protein on apical membranes of rat kidney nephron segments

Abstract

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR1.2) have been identified in kindreds with neonatal Bartter’s syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252–391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34–49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluorescence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H+-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+secretory channel in the rat distal nephron.

pH Dependence of the Inwardly Rectifying Potassium Channel, Kir5.1, and Localization in Renal Tubular Epithelia

“…One such example is thought to be the pH-dependent regulation of the renal secretory K ϩ channel, Kir1.1. This channel is found in the apical membrane of the tubular epithelia (15), where it is inhibited by intracellular, but not extracellular, acidification with a pK of 6.8 (16). Mutations in Kir1.1 that alter its pH sensitivity are responsible for inherited forms of antenatal Bartter's syndrome (17).…”

Mosaic expression of claudins in thick ascending limbs of Henle results in spatial separation of paracellular Na + and Mg 2+ transport

“…It has been previously shown that ammonium transport and the expression of ROMK1, an NKCC2 splice variant, and cldn19 are heterogeneous in the TAL (19,(35)(36)(37). The first description of different TAL cell types was in 1967 by Allen and Tisher (38), who discovered two cell types in rat TAL by means of scanning electron microscopy: R (rough) cells with prominent microvilli and extensive lateral interdigitations and S (smooth) cells generally devoid of extensive microvilli and with less complex lateral interdigitations.…”

The sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and blood pressure regulation

“…The potassium that enters the cell via this pump is recycled by basolateral potassium transporters. In addition, potassium may also be secreted apically by the renal outer medullary potassium channel (ROMK) and a potassium chloride cotransporter [133]. In addition, NCC modulates transcellular magnesium and calcium reabsorption in the DCT through interaction with the transporters TRPM6 and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 TRVP5, respectively (Figure 1) [16,85].…”