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Articles in PresS, published online ahead of print April 16, 2002
Am J Physiol Renal Physiol, 10.1152/ajprenal.00378.2001
Submitted on December 28, 2001
Accepted on April 9, 2002
1 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA
2 Department of Biomedical Science, University of Sheffield, Shefield, S102TN, United Kingdom
3 Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA
4 Department of Physiology, University of Maryland, Baltimore, Maryland, USA
* To whom correspondence should be addressed. E-mail: Chou-Long.Huang{at}UTSouthwestern.edu.
ROMK channels are present in the cortical collecting ducts (CCDs) of kidney and are responsible for K+ secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K+ secretion in CCDs. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed in Xenopus oocytes and cultured Madin-Darby canine kidney (MDCK) cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ~6 hrs. Coexpression with the Lys-44
Ala (K44A) dominant-negative mutant dynamin, but not wild type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn-371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of K44A dynamin mutant on endocytosis of the channel. Co-immunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in MDCK cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.
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