Evidence for the binding of calmodulin to endogenous B-50 (GAP-43) in native synaptosomal plasma membranes

The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.