Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na⁺ + K⁺)-ATPase, Mg²⁺-ATPase, and (Ca²⁺ + Mg²⁺)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca²⁺ fluxes via the Na⁺/Ca²⁺ exchanger, before and after modification of an imposed Na⁺ gradient by penetrating or nonpenetrating Na⁺ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na⁺ + K ⁺)-ATPase, basal Mg²⁺-ATPase, and (Ca²⁺ + Mg ²⁺)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the V_max of the latter activity, the K_m for Ca²⁺ (0.13 μM; the same as that for Ca²⁺-pumping) being unaltered by saponin. An increment of 20.5% in the V_max of (Ca²⁺ + Mg²⁺)-ATPase activity with 10 μM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca²⁺ gradient. Thus, the saponin-induced increment in (Ca²⁺ + Mg²⁺)-ATPase due only to exposure of occluded sites (as opposed to Ca²⁺ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na⁺-channel opening measured indirectly by dissipation of an imposed Na⁺ gradient utilized to drive a large ⁴⁵Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na⁺ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.