This proposal's long-term objective is to characterize the proteins involved in organic molecule transport across insect cell membranes. Membrane protein mediated neurotransmitter, amino acid, and sugar transport systems are essential for insect homeostasis. In contrast to glucose and amino acid transport in bacteria, in yeast and in mammals, our knowledge of these transport processes in insects is rudimentary. More recently plasma membrane neurotransmitter transporters have been identified and cloned in vertebrates. We have preliminary information that similar neurotransmitter transporters, some of which appear to be unique, occur in insects. The specific objectives of this current proposal are to characterize plasma membrane and vesicular neurotransmitter transporters in the lepidopteran, Manduca sexta. M. sexta is being used as a model insect species because its nervous system is much better defined than that of mosquitoes. Once the M. sexta neurotransmitter systems are characterized these probes could be used to identify mosquito neurotransmitter transport. Significant amino acid homology exists between various vertebrate plasma membrane neurotransmitter transporters. Utilizing this fact oligonucleotide primers will be made using the conserved domains as templates for primer design. The primers will then be used for the polymerase chain reaction (PCR) amplification of the putative M. sexta plasma membrane neurotransmitter transporter. These PCR products or alternatively rat plasma membrane and vesicular neurotransmitter transporters will be used for screening of M. sexta cDNA libraries.. However, if this strategy fails, alternative protocols, ie. expression cloning of the transporters will be attempted. Once the cDNA are isolated, they will be expressed and their function and pharmacology determined using established methods. Characterization of these transporter proteins will facilitate further research on transmitter recycling in the nervous system in general, and in the insect brain in particular. Further, the proposed studies will aid more precise determination of transmitter distribution in the insect central nervous system. Such knowledge may enable us to target these transporters as a site for manipulating insect populations. The control of insects, in particular mosquitoes, is critical for the disruption of many mosquito borne diseases of man.
