Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte.
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.
Tanemoto
M
M
First Division of Internal Medicine, University of Tokyo, Tokyo 113, Japan.
Vanoye
C G
CG
Dong
K
K
Welch
R
R
Abe
T
T
Hebert
S C
SC
Xu
J Z
JZ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D005905:Glyburide / Q000494:pharmacology*; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D064233:Sulfonylurea Receptors; D014158:Transcription, Genetic; D014982:Xenopus laevis
D018528:ATP-Binding Cassette Transporters; C577508:Abcc8 protein, mouse; C577509:Abcc8 protein, rat; C577511:Abcc9 protein, mouse; C510693:Kcnj1 protein, mouse; C510692:Kcnj1 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D005905:Glyburide