LOCALIZATION OF SODIUM CALCIUM EXCHANGER & SARCO/ENDOPLASMIC RETICULUM CALCIUM PUMP IN PORCINE CORONARY ARTERY SMOOTH MUSCLE

Abstract

<p>Calcium (Ca2+) is an important signaling molecule hence its movement across cell membranes must be tightly regulated. Ca2+ transporters play a key role in this regulation. The sarco/endoplasmic reticulum (SER) Ca2+ pump (SERCA) and plasma membrane Ca2+ pump (PMCA) keep [Ca2+]i levels low. The sodium calcium exchanger (NCX) may work to expel Ca2+ or bring it into the cell. NCX has been proposed to playa role in refilling the SER Ca2+ pool. We recently reported a functional linkage between CXl and SERCA2 in smooth musc le cells cultured from pig coronary artery. The membrane invaginations termed caveolae (lipid rafts containing caveolin) may also playa role in directing Ca2+ movements during cell signalling. The overall objective of this thesis was to determine whether NCX, SERCA and caveolin proteins were spatially linked (colocalized) in pig coronary artery smooth muscle. This was investigated by examining their co-migration in detergent treated microsomal membranes upon sucrose density gradient fractionation. The fractions were analyzed for the abundance of NCX1, SERCA2, caveolin-1 , lipid raft markers, and cytoskeletal proteins. Then, the Pearson's and Spearman' s correlation coefficient between each proteins distribution was determined. The results indicate there was a significant correlation in the distribution of NCXl and SERCA2, NCXl and caveolin-1 , and SERCA2 and caveolin-1 in the flotation; however the migration was not perfect. In conclusion, NCXl and SERCA2 co-migrate to similar types of membrane domains in pig coronary artery smooth muscle. These domains are rich in lipid rafts and include caveolae. The results also indicate that although a spatial interaction exists, co-localization may not always be present. Thus, CXl may contribute to the SER refilling but it may not be solely responsible for this process. The partial association of NCXl and caveolin-1 suggests the role of caveolae in some Ca2+ signalling pathways but not others.</p>