James J. Chambers, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The purpose of the proposed set of experiments is to examine the intracellular N-terminal domain of the HERG voltage-gated potassium channel. A portion of the N-terminal region is structurally homologous to PAS Domains - ubiquitous structural motifs involved in sensory transduction that are found in a variety of sensory proteins. A number of PAS domains have been found to be associated with bound cofactors through which these PAS domain-containing proteins detect changes in environmental variables and effect signaling change. Two main approaches will be used to discover molecules that affect the HERG PAS domain, an experimental approach that borrows from the disciplines of molecular biology, biochemistry, and NMR, as well as a theoretical approach involving computational protein study and virtual library screening. Lead compounds that are hypothesized to affect the PAS domain derived from any aspects of this project will be experimentally examined for their ability to alter the biophysics of functional HERG channels heterologously expressed in Xenopus oocytes. [unreadable] [unreadable]

Walking and swimming are produced by the coordinated activity of brain cells called neurons that communicate with each other using chemical signals known as neurotransmitters. The chemical signals are passed from one neuron to another across a network of neurons in the brain, then on to neurons in the spinal cord, and finally, the signal is passed to muscles. Some neurotransmitters increase neuron activity, whereas others decrease neuron activity. It is not known how different neurotransmitters balance their effects to coordinate neuron network activity and enable normal locomotion. The goal of this project is to investigate the role of one particular neurotransmitter in the brain to better understand how it regulates locomotion. The project will be carried out using zebrafish because this vertebrate animal has a more simple brain and spinal cord organization than mammals, and it is transparent, which makes it easy to examine under a microscope. Due to the many similarities in neurotransmitters and locomotor networks between zebrafish and mammals, this work can have wide-reaching implications. This project is a collaboration between four faculty researchers from three colleges. Undergraduates at each institution will participate in the research and have access to the expertise across institutions. The research team, including faculty, graduate and undergraduate students, will perform teaching demonstrations about neuroscience at nearby middle and high schools, and students from these schools will visit the investigators' laboratories. Through this outreach effort, 300-600 primarily underrepresented minority middle and high school students will be exposed to scientists and neuroscience.

The central hypothesis that will be tested in this project is that, during early larval stages, a limited number of gamma amino butyric acid (GABA) type A receptor isoforms regulate locomotor networks through a small number of hindbrain reticulospinal neurons. To test this hypothesis, the expression of GABA-A receptor subunits will be determined, the effects of genetic inactivation of GABA-A receptor subunits on locomotor network activity and behavior will be investigated, and the contribution of select hindbrain neurons in generating GABA-A receptor-mediated hyperactive behavior will be assessed. These studies will be carried out in genetically modified zebrafish using microscopy, electrophysiological recordings, quantitative analysis of locomotor behavior, and a novel photochemical approach that enables control of specific GABA-A receptors using different wavelengths of light.