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Am J Physiol Renal Physiol (August 21, 2002). doi:10.1152/ajprenal.00161.2002
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Articles in PresS, published online ahead of print August 21, 2002
Am J Physiol Renal Physiol, 10.1152/ajprenal.00161.2002
Submitted on April 26, 2002
Accepted on August 5, 2002

Characterization of plasma membrane Ca2+ ATPases and their contribution to transcellular Ca2+ flux in MDCK epithelial cells

Sertac N. Kip1 and Emanuel E. Strehler1*

1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

* To whom correspondence should be addressed. E-mail: strehler.emanuel{at}mayo.edu.

Plasma membrane calcium ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using reverse-transcriptase PCR, we detected PMCA1b, PMCA2b (rare) and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na+/Ca2+ exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring 45Ca2+ flux across MDCK cell monolayers indicated that two thirds of apico-basolateral Ca2+ transport were provided by NCX and one third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, trifluoperazine). The importance of PMCA4b for basolateral Ca2+ efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apico-basolateral Ca2+ transport to about 140% while antisense treatment reduced Ca2+ flux approximately 45% compared to controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca2+ reabsorption in the distal kidney.




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