Characterization of PMCA isoforms and their contribution to transcellular Ca2+ flux in MDCK cells.
Plasma membrane Ca(2+) ATPases (PMCAs) are ubiquitous in Ca(2+)-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(+)/Ca(2+) exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring (45)Ca(2+) flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca(2+) transport was provided by Na(+)/Ca(2+) exchanger and one-third by PMCAs, as determined in Na(+)-free media and using various PMCA inhibitors (La(3+), vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca(2+) efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca(2+) transport to approximately 140%, whereas antisense treatment reduced Ca(2+) flux approximately 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 Ca(2+) reabsorption in the distal kidney.
Kip
Sertac N
SN
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Strehler
Emanuel E
EE
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D002478:Cells, Cultured; D004285:Dogs; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D007536:Isomerism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases; D012333:RNA, Messenger / Q000032:analysis; D019831:Sodium-Calcium Exchanger / Q000235:genetics; D014162:Transfection
D027682:Cation Transport Proteins; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium