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+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM 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.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000032:analysis; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012503:Saponins / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013570:Synaptic Membranes / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology
D012503:Saponins; D000001:Calcimycin; D013779:Tetrodotoxin; D012964:Sodium; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium