Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevisoocyte

Abstract

Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive K(+) channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic beta-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba(2+)-sensitive K(+) currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K(+) channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K(+) channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH(2) terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.

Sulfonylurea-Sensitive K+ Transport is Involved in Cl− Secretion and Cyst Growth by Cultured ADPKD Cells

“…Similarly, coinjection of K ir 1.1a (ROMK1) and CFTR cRNA into Xenopus oocytes led to the expression of K ϩ -selective channels with sulfonylurea sensitivity and ATP-gating properties (19). Other binding cassette proteins, particularly the SUR2 forms, may also associate with ROMK1 or ROMK2 in vivo (29). It is possible that CFTR may control such channels in the apical membranes of ADPKD cells in a manner similar to its control of the epithelial Na channel (ENaC).…”

Hemoglobin Is Expressed by Mesangial Cells and Reduces Oxidant Stress

“…31 In addition to the glomeruli, the following nephron tubular segments were microdissected: Proximal convoluted tubules; proximal straight tubules; medullary and cortical thick ascending limb of Henle's loop; distal convoluted tubules; and cortical, outer medullary, and inner medullary collecting ducts.…”

CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 potassium channels in mouse kidney

“…In addition, CFTR-ROMK functional interaction can be regulated by cAMP-dependent protein kinase A (PKA), since increasing cAMP abrogates the effect of CFTR on glibenclamide sensitivity of ROMK in Xenopus laevis oocytes (21). However, other ABC proteins, the sulfonylurea receptors (SUR1/2), have been implicated in the effect of glibenclamide on ROMK (25) as well as ATP and glibenclamide sensitivities of the other ATP-sensitive inward rectifier channels (K ATP , Kir6.x; refs. 12,13,26).…”