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J. Biol. Chem., Vol. 276, Issue 47, 44347-44353, November 23, 2001
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From the ATP-regulated
(KATP) channels are formed by an inward rectifier
pore-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family
(sulfonylurea receptor (SUR) or cystic fibrosis transmembrane
conductance regulator). The latter is required to confer glibenclamide
sensitivity to KATP channels. In the mammalian kidney
ROMK1-3 are components of KATP channels that mediate
K+ secretion into urine. ROMK1 and ROMK3 splice variants
share the core polypeptide of ROMK2 but also have distinct
NH2-terminal extensions of 19 and 26 amino acids,
respectively. The SUR2B is also expressed in rat kidney tubules and may
combine with Kir.1 to form renal KATP channels. Our
previous studies showed that co-expression of ROMK2, but not ROMK1 or
ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive
K+ currents. These data suggest that the
NH2-terminal extensions in both ROMK1 and ROMK3 block
ROMK-SUR2B interaction. Seven amino acids in the
NH2-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH2-terminal deletion and substitution mutants and
examined glibenclamide-sensitive K+ currents in oocytes
when co-expressed with SUR2B. These studies identified an amino acid
triplet "IRA" within the conserved segment in the NH2
terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to confer
glibenclamide sensitivity to the expressed K+ currents. The
position of this triplet in the ROMK1 NH2-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation studies with
hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct
interaction between these two proteins is required for glibenclamide
sensitivity of induced K+ currents in oocytes. These
results suggest that the IRA triplet in the NH2-terminal
extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly
of the renal secretary KATP channel.
An Amino Acid Triplet in the NH2 Terminus of Rat
ROMK1 Determines Interaction with SUR2B*
§,
,,, and§§§
Department of Cellular and Molecular
Physiology, Yale University School of Medicine, New Haven, Connecticut
06520-8026 and the Divisions of ** Genetic Medicine and
¶ Nephrology, Vanderbilt University Medical Center, Nashville,
Tennessee 37232
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by National Institutes of Health Grant DK54998.
Present address: IDEXX Laboratories, Inc., One IDEXX Dr.,
Westbrook, ME 04092.
§§
To whom correspondence should be addressed. Tel.: 203-785-6696;
Fax: 203-785-7678; E-mail: steven.hebert@yale.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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