Evidence for glial sodium-calcium exchanger 1 association with caveolin-1 and 2

초록

The Na+-Ca2+ exchanger (NCX) is involved in regulation of intracellular calcium concentration via the forward mode (Ca2+ extrusion) or the reverse mode (Ca2+ influx). Because they play a role in cellular calcium homeostasis, NCX could be an important regulator for signaling factors. For many types of cells, the signaling factors are now known to be concentrated in microdomains called caveolae. Caveolins (CAVs) are component proteins of caveolae. There is a report that NCX1 associated caveoin-3 in cardiac sarcolemmal vesicles. In this experiment, we investigate the interaction between NCX 1 and caveolin proteins in caveolin-3 null cells, C6 glioma cells using RT-PCR and western blotting technique. When the mRNA and protein expression of CAV-1, -2 and 3 was investigated in C6 glioma cells, CAV-1 and 2 was expressed high level in both mRNA and protein level. In contrast, no the expression of mRNA and protein of CAV-3 was observed. Different with caveolin, mRNA expression of NCX1, 2 and 3 was observed. Using the gradient fractionated glial membrane, the peaks of NCX1, CAV-1 and CAV-2 proteins co-localize more closely in fractions 4 and 5. In order to confirm the association of NCX1 with caveolin, immunoprecipitaion experiments were performed. The presence of NCX1, CAV-1 and CAV-2 was detected following precipitation with NCX1 antibody. In addition, the presences of CAV-1 and NCX1 and CAV-2 and NCX were detected following precipitation with each CAV-1 and CAV-2 antibody, respectively. Cholesterol chelation in C6 glioma cell decreased NCX1 activity. From these results, in the case of lack of CAV-3, NCX1 bind CAV-1 and/or CAV-2. It suggests that all types of caveolin might act as signaling factor of NCX1.