This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Reductionist analyses of the molecular and cellular basis of learning and memory are particularly important in understanding the neural functional design of normal human memory, as well as in age related deficits and pathological states, for example, Alzheimer's, Parkinson's and Huntington's diseases. A number of invertebrate models including Aplysia californica, Drosophila melanogaster, and Caenorhabditis elegans have helped define some of the important biological substrates of behavior. In these species and others, ion channels have been shown to play important molecular roles in various neural processes including simple (e.g. mechanosensation) to complex behavior (e.g. learning and memory). Learning, which may be defined as neural plasticity in response to environmental signals, involves specific changes at synapses. The unique characteristics associated with the ion channels present in the synaptic membrane make them pivotal in understanding the basis of behavior and the plasticity of the nervous system. In particular, ion channels linked to different glutamate receptors are known to be involved in synapse strengthening and long term potentiation. Recently, behavioral neurobiologists have been focusing attention on another class of ion channels, the acid sensing ion channels (ASICs). These channels are activated by extra-cellular hydrogen ions, whose concentration is increased in the synaptic cleft upon neurotransmitter release. It is proposed that ASICs conduct cations and help regulate neurotransmitter release. These channels represent a subclass of the degenerin/epithelial Na+ channel (DEG/ENaC) superfamily of ion channels that are present in all examined multicellular eukaryotes and are most well-characterized in C. elegans. Members of the DEG/ENaC superfamily from various species show similarity in terms of their sequence and predicted topology with each individual DEG/ENaC subunit consisting of two transmembrane domains and a large extra-cellular domain. The crystal structure of a chicken ASIC has shown that it is made of three identical subunits, where there is some evidence to indicate that other ASICs may be formed of heteromeric subunits. C. elegans, a microscopic nematode, is a well-studied model for neural network, bioimaging and genomic studies. The almost wiring of its 302 neurons is known and the complete genome of this transparent organism has been sequenced. Information from studies on the larger Deg/ENaC gene superfamily in C. elegans, makes this organism ideal for studying the structural and functional complexity of ASICs. In C. elegans genome, the DEG/ENaC superfamily comprises of 28 members, of which 7 have been characterized genetically and are implicated in mechanosensation, proprioception, and in the regulation of ultradian rhythms. The focus of this proposal is to characterize del-1, a C. elegans gene whose product is very similar in sequence to that of asic-1. Specifically, we propose to investigate the expression of del-4 using fluorescent tags, and test its potential interactions with asic-1 using fluorescence energy transfer bioimaging techniques. We have already initiated behavioral testing of a del-4 deletion mutant and our encouraging preliminary results are presented in the proposal. We expect that information obtained from using the C. elegans model will open new avenues to understanding the role of ASICs in higher organisms and provide foundations for understanding human neurological disorders. During the course of carrying out experimentation required to accomplish the goals listed in our proposal, we plan to train undergraduate and graduate students at Delaware State University. We will also build additional capabilities in terms of carrying out sophisticated molecular biology work and develop our bioimaging facilities, while coordinating with other institutes in the state of Delaware including the core facilities available at DBI. Accomplishing the scientific and infrastructural goals will provide our group the necessary competitiveness for writing a proposal for direct federal funding by the end of year two of the proposal. The PI and the co-PI will work closely with the mentor Dr. J. Rosen from University of Delaware, for scientific discussions and guidance towards preparing a competitive NIH R01 proposal. We will also have regular electronic contact with Dr. N. Tavernarakis at the Institute of Molecular Biology and Biotechnology, whose lab is focusing on asic-1, t discuss scientific and technical issues so as to continually monitor the direction of our research.
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