Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a.
Sarco(endo)plasmic reticulum Ca2+ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca2+ uptake, Ca2+-Mg2+-ATPase, Ca2+-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca2+ uptake, Ca2+-Mg2+-ATPase, and Ca2+-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress.
Dept. of Medicine, HSC 4N41, McMaster Univ., 1200 Main St. West, Hamilton, Ontario L8N 3Z5 Canada. groverak@mcmaster.ca
Ashok K
AK
Grover
Chiu-Yin
CY
Kwan
Sue E
SE
Samson
D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000187:drug effects*; D002461:Cell Line, Transformed; D004721:Endoplasmic Reticulum / Q000187:drug effects; D006801:Humans; D008861:Microsomes / Q000187:drug effects; D018384:Oxidative Stress / Q000502:physiology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D020033:Protein Isoforms / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D020033:Protein Isoforms; D030421:Peroxynitrous Acid; D017301:Ca(2+) Mg(2+)-ATPase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506943:ATP2A1 protein, human; C506946:ATP2A2 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium