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. The first project, completed in collaboration with David Siderovski and Francis Willard (University of North Carolina), involved characterization of a mutation that disrupted GoLoco motif protein binding to heterotrimeric G-proteins. Both classes of proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Gi) of Galpha subunits, and thus it is assumed that a Gi-GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gi subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity (""""""""GLi"""""""") mutation prevented Gi1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gi1 subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbeta/gamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gi subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gi1-GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction. Willard et al., J. Biol. Chem. 283:36698-36710, 2008. A second project involved development of a reduced model system to study endocannabinoid mobilization, transport, and signaling. Endocannabinoids (eCB) such as 2-arachidonylglycerol (2 AG) are lipid metabolites that are synthesized in a postsynaptic neurons and act upon CB1 cannabinoid receptors (CB1R) in presynaptic nerve terminals. This retrograde transmission underlies several forms of short and long term synaptic plasticity within the central nervous system. In this study, we constructed a model system based on isolated rat sympathetic neurons in which an eCB signaling cascade could be studied in a reduced, spatially compact, and genetically malleable system. We constructed a complete eCB production/mobilization pathway by sequential addition of molecular components. Heterologous expression of four components were required for eCB production and detection: metabotropic glutamate receptor 5a (mGluR5a), Homer 2b, diacylglycerol lipase α, and CB1R. In these neurons, application of L-glutamate produced voltage-dependent modulation of N-type Ca2+ channels mediated by activation of CB1R. Using both molecular dissection and pharmacological agents, we provide evidence that activation of mGluR5a results in rapid enzymatic production of 2 AG followed by activation of CB1R. These experiments define the critical elements required to recapitulate retrograde eCB production and signaling in a single peripheral neuron. Moreover, production/mobilization of eCB can be detected on a physiologically relevant time scale using electrophysiological techniques. The system provides a platform for testing candidate molecules underlying facilitation of eCB transport across the plasma membrane. Won, Y-J, Puhl, H.L., and Ikeda, S.R., Journal of Neuroscience, in press, 2009. The third project explored a novel methodology to target specific proteins for modification or ablation on a rapid time scale in living mammalian cells. The system consists of an inducible NIa protease from the tobacco etch virus (TEVp) and a chosen protein into which a TEVp substrate recognition sequence (TRS) was inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a fluorescence resonance energy transfect (FRET)-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 C proceeded exponentially with a time constant of approximately 150 minutes. Two strategies were used to improve cleavage efficiency: 1) The relative concentration of the TEVp fragments to each other was increased 2) The relative concentration of TEVp to the substrate was increased. Both these approaches were hampered by substantial background activity that was attributed to inherent affinity between the TEVp fragments, and demonstrated that TEVp activity was surprisingly insensitive to insertion of large protein domains at the split site. A second TEVp assay, based on changes in inactivation of a modified KV3.4 channel, showed that functional properties of a channel can be using altered using an inducible TEVp system. Similar levels of background activity and variability were observed in both electrophysiological and FRET assays. The results suggested that an optimum level of TEVp expression leading to sufficient inducible activity (with minimal background activity) exists but the variability in expression levels between cells makes the present system rather impractical for single cell experiments. Williams, D.J., Puhl, H.L., and Ikeda, S.R. submitted, 2009. A fourth project, continuing earlier work on the identification of the voltage-gated sodium channel NaV1.8 promoter region (Puhl, H.L. and Ikeda, S.R., J. Neurochem. 106:1209-1124, 2008), was also advanced during this period. With the help of Dr. Rui Costa, the identity of the previously defined region has been confirmed by constructing a transgenic mouse expressing the jellyfish green fluorescence protein driven by a 4 kb segment of genomic DNA. Preliminary results indicate specific expression in primary sensory neurons presumably involved in nociceptive function. The animals establish the importance of the 4 Kb region as a means of expressing proteins specifically in a subset of sensory neurons a finding with potential therapeutic potential. Moreover, the mice provide a means for identifying novel repressive elements that specify sensory neuron expression. Finally, the mice might provide a useful tool for investigating ectopic expression of Nav1.8 in disorders such as multiple sclerosis and ethanol induced cerebellar damage.
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