Annexin V-mediated calcium flux across membranes is dependent on the lipid composition: Implications for cartilage mineralization

Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca²⁺ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca²⁺ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca²⁺ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca²⁺ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca²⁺ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca²⁺ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca²⁺-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca²⁺ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca²⁺ influx, intralumenal crystal growth, and cartilage matrix mineralization.