The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. There are four main projected currently underway. Dr. Huanmian Chen is studying the effect of the RGK class of small GTPase proteins on N-type calcium channels in sympathetic neurons. The RGK family, which includes the proteins Gem, Rad, Rem1 and Rem2, have recently been found to interact with the beta accessory subunit of high voltage-activated calcium channels. Twenty four hours following expression of Rem2, an RGK protein expressed in the nervous system, the function of N-type calcium channels in sympathetic neurons is ablated. Future studies are aimed at: 1) determining the mechanism of channel ablation; 2) establishing the physiological role of channel modulation; and 3) determining the molecular domains of Rem2 that convey this activity. In regard to the latter Aim, a current goal is to utilize the properties of Rem2 to identify specific roles of various high voltage-activated channels in synaptic function. Additionally, collaborative efforts with the laboratory of Dr. Kathy Kelly (NCI) have established that the GTP-binding domain of Gem, another member of the RGK family, is required for calcium channel activity. A second project involves using a technique, bimolecular fluorescent complementation (BiFC), to label specific dimers of G-protein beta/gamma subunits. An eventual goal of this research is to study the trafficking of identified dimers to specific compartments within neurons. Dr. Juan Guo is studying the effects of endocannibinoids on CB1 cannabinoid receptors heterologously expressed in sympathetic neurons. CB1 receptors have recently been implicated in a number of addictive disorders and represent a new therapeutic target for the treatment of alcoholism. In a recently completed study. Dr. Guo demonstrated that several putative endocannabinoids modulate N-type calcium channels via CB1 receptors expressed in sympathetic neurons. The study is the first to show that 2- arachidonoylglycerol and noladin ether are capable of producing voltage-dependent modulation of N-type calcium channels. In addition, it was shown that anandamide produces a CB1 receptor-independent modulation that is voltage-independent and thus distinct from the aforementioned receptor-mediated modulation. Current plan include developing an biosensor based on the coupling of CB1 receptors to GIRK-type potassium channels to detect the release of endocanniboinds in real-time and tagging CB1 receptors with fluorescent proteins to study receptor dimerization using fluorescence resonance energy transfer (FRET) based techniques. Dr. John Partridge, a recent addition to the Section, is investigating the effects of a phosducin and phosducin-like protein on G-protein coupled receptor (GPCR)-mediated modulation of N-type channels. Phosducin and phosducin-like protein bind to G-protein beta/gamma subunits and disrupt signaling between GPCRs and effectors. The latter protein has also been shown to be up-regulated in neuroblastoma cells treated with ethanol. Progress so far indicates that expression of these proteins attenuates GPCR-mediated ion channel modulation in a time-dependent fashion. As the crystal structure of phosducin complexed with G-protein beta/gamma subunits suggests a conversion of phosducin from and an extended to a compact form, Dr. Partridge is attempting to develop a fluorescent biosensor based on these molecules that will detect the initial step of G-protein activation, i.e., the release of the Gbeta/gamma subunit. The current strategy involves tagging phosducin or phosducin-like protein at both termini with fluorescent proteins or fragments of fluorescent proteins. Binding of G-protein beta/gamma subunits should bring the termini of the proteins into apposition thus allowing either FRET or BiFC to be used as a method of detection. The principle motivation behind these studies is to develop a system that would allow universal high throughput detection of ligands and GPCRs, subcellular localization of GPCR activation, and real-time kinetic analysis of G-protein activation. Dr. Henry Puhl, as well as providing molecular biological expertise for our laboratory and several others, is principally involved with dissecting out the genomic elements that convey sensory neuron specificity to the expression of the TTX-resistant sodium channel Nav1.8 encoded by the Scn10a gene. So far two regions of upstream genomic sequence that impacts sensory neuron specific expression have been putatively identified. Using deletional analysis and mouse dorsal root ganglion neurons as reporter hosts, Dr. Puhl has identified a region of the Scn10a promoter that is neuron-specific and a second region that appears to convey sensory neuron specificity. The latter region may represent a novel repressor element. Future studies aim to refine these regions using luciferase-based reported assays in N1E-115 neuroblastoma cells and dorsal root ganglion neurons. Confirmation of promoter sequence will be accomplished by producing transgenic mice expressing green fluorescent protein driven by the identified promoter region. Finally, progress has been made in developing a total internal reflectance fluorescence (TIRF) microscope capable of analyzing FRET-based protein-protein interactions in the membranes of neurons. Fluorescence excitation is provided by HeCd and Argon ion lasers. The individual laser lines are selected and modulated by an acoustical optical tunable filter (AOTF) driven by software designed in the laboratory. Dual emission imaging is accomplished using an image splitter with appropriate emissions filters and a dichroic mirror. Acquired images are analyzed for FRET using custom software currently under development.
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