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 ⁴²Ca²⁺ 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 ∼140%, whereas antisense treatment reduced Ca²⁺ flux ∼45% 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.