Some Molecular Properties of the Synaptic Plasma Membrane Na+‐Ca2+ Exchanger a

The identification of the brain Na+-Ca2+ exchanger protein has not yet been resolved. We have developed a procedure that involves the use of sequential gel filtration, ion exchange, and immobilized metal affinity chromatography in the purification of exchanger activity associated with two protein bands on SDS-PAGE. I The two protein bands have estimated molecular sizes of 36 and 50 kDa, considerably smaller than the proteins isolated from sarcolemmal (70, 120, and 160 kDa)2 and rod outer segment (ROS) membranes (21 5-220 kDa).3s4 Polyclonal antibodies (Abs) were raised against the electroeluted 36-kDa protein, and these Abs cross-reacted with the electroeluted 50-kDa protein on ELISAs, suggesting that the two proteins are closely related or that one may be an abnormally migrating form of the other. The anti-36-kDa serum immunoextracted > 95% of the Na+-Ca2+ exchange activity in solubilized synaptic membranes while preimmune serum extracted none.' These results indicate that the 36-kDa band is a critical component of the brain Na+-Ca2+ exchanger protein. The addition of many protease inhibitors did not lead to the isolation of higher molecular weight proteins. This does not totally rule out the possibility that these bands represent proteolytic fragments from a larger protein. The exchanger activity present in brain membranes differs significantly from the sarcolemmal exchanger in terms of sensitivity to treatment with proteases. Philipson and colleagues reported that treatment of sarcolemma1 membranes with a variety of proteases led to marked activation (200%) of antiporter a~tivity.~ Similar treatment of synaptic membranes, on the other hand, never stimulated exchanger activity and consistently led to inhibition of activity at higher protease concentrations (FIG. 1). These results may indicate that brain proteases have already activated the exchanger in the brain membranes, preventing further stimulation by exogenous proteases and leading to purification of active proteolytic fragments that associate with each other during the reconstitution step. It is also possible, however, that the 36-kDa brain protein represents a specific isoform of the exchanger that is expressed in high levels in the brain.