Characterization of PMCA isoforms and their contribution to transcellular Ca2+ flux in MDCK cells

Plasma membrane Ca2+ ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using RT-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 Ca-45(2+) flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca2+ transport was provided by Na+/Ca2+ exchanger and one-third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca2+ efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca2+ transport to similar to140%, whereas antisense treatment reduced Ca2+ flux similar to45% compared with 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.