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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI034524-01
Application #
3149439
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1993-07-01
Project End
1998-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Riverside
Department
Type
Schools of Earth Sciences/Natur
DUNS #
City
Riverside
State
CA
Country
United States
Zip Code
92521
Sandhu, Sumandeep K; Ross, Linda S; Gill, Sarjeet S (2002) A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction. Eur J Biochem 269:3934-44
Umesh, Anita; Gill, Sarjeet S (2002) Immunocytochemical localization of a Manduca sexta gamma-aminobutyric acid transporter. J Comp Neurol 448:388-98
Sandhu, Sumandeep K; Ross, Linda S; Gill, Sarjeet S (2002) Molecular cloning and functional expression of a proline transporter from Manduca sexta. Insect Biochem Mol Biol 32:1391-400
Chiu, C; Ross, L S; Cohen, B N et al. (2000) The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling. J Exp Biol 203:3531-46
Pullikuth, A K; Gill, S S (1999) Identification of a Manduca sexta NSF ortholog, a member of the AAA family of ATPases. Gene 240:343-54
Pullikuth, A K; Gill, S S (1997) Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases. Gene 200:163-72
Ross, L S; Gill, S S (1996) Limited growth PCR screening of a plasmid library. Biotechniques 21:382-4, 386
Mbungu, D; Ross, L S; Gill, S S (1995) Cloning, functional expression, and pharmacology of a GABA transporter from Manduca sexta. Arch Biochem Biophys 318:489-97