The magnitude and duration of chemical neurotransmission is determined by reciprocal presynaptic activities of neurotransmitter release and reuptake. At sympathetic synapses of the vertebrate autonomic system, the catecholamine neurotransmitters norepinephrine (NE) and epinephrine (Epi) are rapidly cleared by transporter proteins thought to be enriched in presynaptic terminals and varicosities. The efficient recovery of NE and Epi permits repetitive sympathetic signaling without synaptic receptor desensitization, spatially limits the response to neurotransmitter, and helps maintain presynaptic catecholamine stores necessary for sustained signaling. Alterations in NE transport, as occurs with pharmacologic blockade by cocaine or tricyclic antidepressants, leads to a an augmentation of synaptic responses, a spillover of NE out of the synapse, the triggering of extrasynaptic receptors, and subsequent receptor desensitization. Whereas the kinetic behavior and drug sensitivities of catecholamine transporters are well described, molecular details of transporter structure, localization, and regulation have been unavaIlable. Recently, we have cloned the first cDNAs encoding cocaine and antidepressant-sensitive NE transporters (NETs) from the human medulloblastoma SK-N-SH, characterized the functional properties of expressed carriers in transfected cells, and developed NET specific antibodies for biochemical and immunocytochemical studies. We now propose to 1) identify and dissect homologous NETs and Epi transporters (ETs) expressed in the vertebrate heart, 2) elucidate molecular mechanisms for acute NET regulation using radiotracer flux and ligand binding, single cell electrophysiological techniques, and biochemical analysis of modified NET protein and 3) determine where and how transporters are spatially localized to presynaptic terminals using autoradiography and immunocytochemistry. To accomplish these objectives, we will perform experiments on individual gene products in reconstituted systems as well as primary tissues expressing NETs. Data from these studies should substantially augment our understanding of presynaptic control mechanisms involved in sympathetic neurotransmission.
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