Ca(2+) and Mg(2+)/ATP independently trigger homotypic membrane fusion in gastric secretory membranes

Exocytic activation of gastric parietal cells represents a massive transformation. We studied a step in this process, homotypic fusion of H,K-ATPase-containing tubulovesicles, using R18 dequenching. Ca²⁺ and Mg²⁺/ATP each caused dramatic dequenching, reflecting a change in R18 distribution from 5% to 65–90% of the assay's membranes in 2.5 min. These stimuli also triggered fusion between tubulovesicles and liposomes. Independent confirmation that dequenching represented membrane fusion was established by separating tubulovesicle–liposome fusion products on density gradients. Only agents that trigger fusion allowed the transmembrane H,K-ATPase to move to low-density fractions along with R18. EC₅₀ for Ca²⁺-triggered fusion was 150 n m and for Mg²⁺/ATP-triggered fusion 1 m m , the latter having a Hill coefficient of 2.5. ATP-triggered fusion was specific for Mg²⁺/ATP, required ATP hydrolysis, and was insensitive to inhibition of NSF and/or H,K-ATPase. Fusion initiated by either trigger caused tubulovesicles to become resistant to subsequent challenge by either trigger. Ca²⁺-and Mg²⁺/ATP-triggered fusion required protein component(s) in tubulovesicles, though this was required in only one of the fusing membranes since tubulovesicles fused well with liposomes containing no proteins. Our data suggest that exocytosis in parietal cells is triggered by separate but interacting pathways and is regulated by self-inhibition.