Monoamine transporters transport cycle

Interestingly, we have recently identified a mutation of a tyrosine in the third intracellular loop of the hDAT that causes a major alteration in the conformational equilibrium of the transport cycle, and thus as such is comparable to mutants on G protein-coupled receptors causing constitutive isomerization of the receptor to the active state (66). Most importantly, this conclusion is based on the observation that mutation of the tyrosine completely reverts the effect of Zn2+ at the endogenous Zn2+ binding site in the hDAT (50,51) from potent inhibition of transport to potent stimulation of transport (Fig. 6). In the absence of Zn2+, transport capacity is reduced to less than 1% of that observed for the wild-type, however, the presence of Zn2+ in only micromolar concentrations causes a close to 30-fold increase in uptake (66). Moreover, it is found that the apparent affinities for cocaine and several other inhibitors are substantially decreased, whereas the apparent affinities for substrates are markedly increased (66). Notably, the decrease in apparent cocaine affinity was around 150-fold and thus to date the most dramatic alteration in cocaine affinity reported upon mutation of a single residue in the monoamine transporters (66). 

The VMATs are also among the very few vesicular neurotransmitter transporters whose turnover number is known. At 29° C, they transport —5 molecules of serotonin per second and up to 20 molecules of dopamine (Peter et al., 1994). Since synaptic vesicles contain 5 to 20,000 molecules of transmitter and can recycle within at least 20 seconds (Ryan and Smith, 1995 Rizzoli et al., 2003), this rate has important implications for quantal size. At 5 molecules/second, the vesicle would contain only 100 molecules of transmitter after 20 seconds—if there were only one transporter per vesicle. Recent estimates suggest several transporters per vesicle (Takamori et al., 2006), but these might still not suffice to fill a rapidly cycling vesicle with monoamine unless the turnover was substantially higher at 37° C, where it is more difficult to measure transport accurately due to increased membrane leakiness. Indeed, the ability to determine the turnover of VMATs has been enabled by the availability of ligands to quantify the transporter and hence provide a denominator for measurements of transport. 

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