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 projects currently underway. The first examines the effect of RGK family 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 shown to interact with the beta accessory subunit of high voltage-activated calcium channels. Preliminary data indicate that expression of Rem2, an protein expressed primarily in the nervous system, greatly reduces the current flowing through N-type calcium channels in sympathetic neurons. Future studies are aimed at: 1) determining the mechanism of current reduction; 2) establishing a physiological role for Rem2; and 3) determining the molecular domains of Rem2 that convey calcium channel activity. We have recently cloned a novel variant of Rem2 and prepared GFP-tagged and adenoviral constructs to explore the function and distribution of the molecule. In addition, vectors expressing an N-type calcium channel alpha one subunit bearing an external epitope tag have been constructed and are being characterized to determine whether Rem2 alters calcium channel surface expression. The second project explores mechanisms of signaling between heterologously expressed G-protein coupled receptors (GPCR) and calcium and potassium channels in sympathetic neurons. CB1 cannabinoid receptors have been implicated in a number of addictive disorders and represent a new therapeutic target for the treatment of alcoholism. In a recently completed study, we demonstrated that several putative endocannabinoids modulate N-type calcium channels via CB1 receptors expressed in sympathetic neurons. The study was 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. We are currently exploring how another presynaptically-targeted GPCR, mGluR8, modulates ion channels in neurons A third area of investigation focuses on mechanisms by which phosducin and phosducin-like protein influence 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 an extended to a compact form, we are exploring the development of fluorescent biosensors 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 fluorescence resonance energy transfer (FRET) or bi-molecular fluorescence complementation 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. The final project focuses on dissecting out the genomiiic 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, we have 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.
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