EXPRESSION AND LOCALIZATION OF ANNEXIN-VII (SYNEXIN) IN MUSCLE-CELLS

Annexin VII (synexin) is a member of the annexin family of proteins, w hich are characterized by Ca2+-dependent binding to phospholipids. We used PCR to isolate from a lambda gt11-mouse fibroblast library annexi n VII cDNA fragments corresponding to the two isoforms found in both h umans and Dictyostelium discoideum. The two isoforms of 47 kDa and 51 kDa differed by 22 amino acids inserted into the proximal third of the hydrophobic N terminus. Annexin VII-specific polypeptides expressed i n Escherichia coli were used to generate isoform-specific monoclonal a ntibodies. Expression of the two isoforms during myogenesis was follow ed in the myogenic cell lines BC(3)H1 and L6. Only the 47 kDa isoform was present in undifferentiated L6 or BC(3)H1 myoblasts. The 51 kDa is oform appeared after myogenesis had been induced and in striated muscl e only the 51 kDa isoform was observed. Immunofluorescence showed that annexin VII was located in the cytosol of mononucleated and fused pol ynucleated cultured cells, whereas in striated muscle, annexin VII was located preferentially at the plasma membrane and the transverse tubu les. However, there was also some residual cytosolic staining, which w as more abundant in type II (fast twitch) than in type I (slow twitch) fibers. Permeabilization of L6 cells with digitonin in the presence o f 5 mM EGTA led to a release of annexin VII from the cells, which para lleled the loss of cytosolic lactate dehydrogenase (LDH) at low deterg ent concentrations (50 mu M) In the presence of 100 mu M extracellular Ca2+, annexin VII remained bound to the plasma membrane even in the p resence of high digitonin concentrations, Incubation with the Ca2+-spe cific ionophore A23187 and 100 mu M extracellular Ca2+ led to a redist ribution of annexin VII from the cytosol to the plasma membrane after 30 minutes of incubation. The results obtained indicate a developmenta lly and Ca2+-regulated localization and expression of annexin VII and raise the possibility that annexin VII may play a role in excitation-c ontraction coupling in skeletal muscle.