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 were four main areas of progress during the current funding period.? ? Based on the previous results with the RGK protein Gem (Ward et al., Mol Cell Biol. 24:651?661, 2004), we initiated studies on Rem2, the sole member of the RGK family with a predominately neural expression profile (Chen et al., J Neurosci. 25:9762?9772, 2005). We originally cloned Rem2 from rat dorsal root ganglion (DRG) mRNA based on the originally deposited sequence (AF084464). During the course of our experiments, examination of ESTs and genomic DNA sequences from several vertebrate species (while searching for alternative splicing variants) revealed two in-frame ATGs 5' to the start codon assigned in AF084464. We cloned the longest predicted open reading frame (ORF), encoding an additional 69 residues at the N-terminus, from rat DRG mRNA (sequence deposited in GenBank: accession no. AY916790). The ORF assigned in AF084464 likely arose from a sequencing error rather than representing a genuine alternative splice. In the current study, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high voltage-activated calcium channels without affecting low voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C-terminus. Mutation of a putative GTP binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-beta-S, a hydrolysis-resistant GDP analog, via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally radiolabeled omega-conotoxin GVIA cell surface binding to tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when the calcium current was totally abolished. Taken together, the results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel beta-subunits of the preassembled N-type calcium thereby forming a non-conducting species. These data revealed a novel form of voltage-independent calcium channel modulation that may impact plastic changes in synaptic transmission and provide the basis for further experiments exploring mechanism and function of RGK proteins.? ? Phosducin (PDC) and phosducin-like protein (PDCL) 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. We have found that expressing PDC and PDCL in dissociated superior cervical ganglion (SCG) neurons attenuates voltage-dependent inhibition of N-type calcium channels in a time-dependent manner (Partridge et al., Mol Pharmacol. 70:90?100, 2006). Expression of PDC and PDCL attenuated voltage-dependent inhibition of N-type calcium channels, a G-protein beta/gamma-dependent process, in a time-dependent fashion. Calcium current inhibition following acute exposure to norepinephrine was minimally altered by PDC or PDCL expression. However, in the continued presence of norepinephrine, PDC or PDCL relieved calcium channel inhibition when compared with control neurons. We observed similar results following activation of heterologously expressed metabotropic glutamate receptor 2 with L-glutamate. Neurons expressing PDC or PDCL maintained suppression of inhibition following re-exposure to agonist. Unlike other G-protein beta/gamma sequestering proteins that abolish the acute inhibition of calcium channels, PDC and PDCL require prolonged agonist exposure before effects on modulation are realized.? ? Fluorophore-assisted light inactivation (FALI) is a technique in which a fluorophore is attached to a targeted protein either directly or indirectly via a labeled intermediate Following illumination, energy is transferred from the fluorophore to oxygen molecules resulting in the generation of reactive oxygen species (ROS) such as singlet oxygen. The ROS reacts with amino acids in close proximity to the fluorophore producing functional inactivation through incompletely understood mechanisms. We examined a novel FALI modality that utilizes a fluorescein conjugated polypeptide, alpha-bungarotoxin (BTX), and a 13 amino acid BTX-binding site engineered into the N-terminus of mGluR8a (Guo et al., J Physiol. 2006 Jul 27; [Epub ahead of print]). The tagged mGluR8a was expressed in rat sympathetic neurons and labeled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of calcium currents using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labeled neurons, mGluR8a-mediated calcium current inhibition was attenuated whereas the holding current and basal calcium currents were only slightly decreased. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signaling pathway, alpha2-adrenergic receptor mediated calcium current modulation, were obtained following illumination of neurons expressing FL-BTX labeled voltage-gated sodium channel beta-2 subunits or ionotropic 5-HT3 receptors?proteins with no overt relationship to GPCR signaling pathways. Second, GPCR-independent calcium current modulation induced by dialyzing neurons with guanylyl imidophosphate from the patch pipette was also affected by FALI. Our results demonstrate that GPCR signaling to N-type calcium channels in neurons was acutely disrupted following FALI. However, collateral effects on non-targeted proteins were also observed. Improving the efficacy of FALI provides a means of spatially restricting effects by allowing lowered expression levels of the tagged protein, decreased illumination power, or the inclusion of quenchers in the reaction. We plan on using fluorophores with improved singlet oxygen quantum yield to increase FALI specificity.? ? Finally, we have developed optical methodology based on Forster resonance energy transfer (FRET) that allows the determination of FRET efficiency and stoichiometry of expressed fluorescent proteins in living cells (Chen et al., Biophys J. 91:L39-41, 2006). Determining these parameters using the three-filter cube approach requires the determination of two constants: 1) the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2) the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.
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