Calmodulin Stimulation of Ca2+‐Dependent ATP Hydrolysis and ATP‐Dependent Ca2+ Transport in Synaptic Membranes

Abstract

We report here characterization of cal-modulin-stimulated Ca²⁺ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis coupled with ATP-dependent Ca²⁺ uptake into membraneous sacs on the cytosolic face of the synap-tosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca²⁺-ATPase and ATP-dependent Ca²⁺ uptake require calmodulin for maximal activity (K_Cm for ATPase = 60 nM; K_Cm for uptake = 50 nM). In the reconstituted membrane system, K_Ca was found to be 0.8 μM for Ca²⁺-ATPase and 0.4 μM for Ca²⁺ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca²⁺ pump in SPM when Ca²⁺ ATPase and Ca²⁺ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca²⁺ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca²⁺ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca²⁺+ Mg²⁺ ATPase as a regulator for cytosolic Ca²⁺.