KINETIC-PARAMETERS FOR THE VESICULAR ACETYLCHOLINE TRANSPORTER - 2 PROTONS ARE EXCHANGED FOR ONE ACETYLCHOLINE

The vesicular acetylcholine transporter (VAChT) mediates ACh storage i n synaptic vesicles by exchanging cytoplasmic ACh with vesicular proto ns. This study sought to determine the stoichiometry of exchange by an alysis of ligand binding and transport kinetics. The effects of differ ent pH values inside and outside, external ACh concentrations, and ele ctrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protoco l. The equilibrium binding of a high-affinity analogue of ACh is inhib ited by protonation with a pK(a) of 7.4 +/- 0.3. A two-proton model fi ts the transport data much better than a one-proton model does, and up take increases at more positive internal electrical potential, as expe cted for the two-proton model. Thus, the results support the two-proto n model. The transport cycle begins with binding of external ACh to ou twardly oriented site 2 (K-ACho = 20 mM) and protonation of inwardly o riented site 1 (pK(a1) = 4.73 +/- 0.05). Loaded VAChT reorients quickl y (73 000 min(-1)) and releases ACh to the inside (K-AChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 b inds a proton (pK(a2) = 7.0), after which VAChT reorients (150 +/- 20 min(-1)) in the rate-limiting step and releases the proton to the outs ide to complete the cycle. Rate constants for the reverse direction al so were estimated. Two protons provide a thermodynamic driving force b eyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh bindi ng site toward cytoplasm, orientational lifetimes, and the rate of non quantal release of ACh from cholinergic nerve terminals, were computer -simulated, and the results are compared with physiological observatio ns.