Drugs of abuse and therapy (e.g., cocaine, amphetamine, fluoxetine) exert their effects by acting on plasma membrane amine transporters. This is evidence that amine transporters function to determine synaptic transmitter levels (and thus the response of pre- and post-synaptic receptors). Recent data suggest that transporter function is regulated. Given the role of amine synapses in drug abuse and neuropsychiatric disease, such-regulation would have significant consequences. The general hypothesis to be tested is: Transporters regulate synaptic transmitter levels by responding similarly to the processes responsible for those transmitter levels. These experiments will determine the factors that regulate transporter trafficking, expression and modulation. Falsifiable predictions will be made from: Hypothesis 1: Expression of transporters occurs by pathways identical to those involved in transmitter secretion. Localization and expression of the transporter will be assessed. Antisense, toxin, and pharmacological strategies will then be used to inactivate secretion components (e.g., cytoskeleton, docking proteins) at specific stages. Hypothesis 2: Modulation of transporter function occurs in response to molecules that affect, or are affected by, synaptic transmitter levels. Transporter expression and trafficking will be assessed in response to signals that affect neurotransmitter release (e.g., Ca2+) or that signal extracellular transmitter levels (e.g., presynaptic receptors). The mechanism of action (e.g., protein synthesis, translocation) will be assessed for each modulator. Hypothesis 3: Leucine motifs in the primary amino acid sequence of the transporter govern targeting to the regulated secretion pathway. Mutant transporters will be produced; their targeting and ability to be regulated will be assessed. Interaction of wild-type and mutant transporters with secretion proteins will be determined. Predictions will be tested using expression of the human serotonin transporter in neurosecretory (PC12) cells. Outcome measures include uptake of radiolabeled substrates, antagonist binding, subcellular fractionation and western blots, and immunomicroscopy. These experiments will 1) elucidate the role of transporters in synaptic signaling; 2) determine the interaction between transmitter secretion and the reuptake process; 3) identify components necessary for transporter expression and regulation; and 4) provide an alternative strategy for regulating levels of amines at the synapse that could be useful in treatments for drug abuse and mental illness.
| Moss, Fraser J; Imoukhuede, P I; Scott, Kimberly et al. (2009) GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET. J Gen Physiol 134:489-521 |
