The primary focus of the section is to further our understanding of the molecular basis of signaling between GTP-binding proteins and voltage-gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. We recently published a study based on the Cre-inducible Rem2 KO mouse developed for our laboratory. Rem2 is the primary RGK protein expressed in the nervous system, but to date, the precise expression patterns of this protein are unknown. In this study, we characterized Rem2 expression in the mouse nervous system. In the central nervous system, Rem2 mRNA was detected in all regions examined, but was enriched in the striatum. An antibody specific for Rem2 was validated using a Rem2 knockout mouse model and used to show abundant expression in striatonigral and striatopallidal medium spiny neurons but not in several interneuron populations. In the peripheral nervous system, Rem2 was abundant in a subpopulation of neurons in the trigeminal and dorsal root ganglia, but was absent in sympathetic neurons of superior cervical ganglia. Under basal conditions, Rem2 was subject to post-translational phosphorylation, likely at multiple residues. Further, Rem2 mRNA and protein expression peaked at postnatal week two, which corresponds to the period of robust neuronal maturation in rodents. This study forms the basis ongoing attempts to elucidate the functions of Rem2 in basal ganglia physiology. Liput DJ, Lu VB, Davis MI, Puhl HL, Ikeda SR. 2016. Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia. Sci Rep 6: 25137. In addition, two methodological manuscripts were published based on our teaching experience over the past 3 years at the Cold Spring Harbor Ion Channels and Synaptic Transmission course: 1) Lu VB, Ikeda SR. 2016. Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels. Cold Spring Harb Protoc 2016: pdb.top087072. G-protein-coupled receptor modulation of voltage-gated ion channels is a common means of fine-tuning the response of channels to changes in membrane potential. Such modulation impacts physiological processes such as synaptic transmission, and hence therapeutic strategies often directly or indirectly target these pathways. As an exemplar of channel modulation, we examine strategies for investigating G-protein modulation of CaV2.2 or N-type voltage-gated clacium channels. We focus on biochemical and genetic tools for defining the molecular mechanisms underlying the various forms of CaV2.2 channel modulation initiated following ligand binding to G-protein-coupled receptors. 2) Lu VB, Ikeda SR. 2016. G-Protein Modulation of Voltage-Gated Ca2+ Channels from Isolated Adult Rat Superior Cervical Ganglion Neurons. Cold Spring Harb Protoc 2016: pdb.prot091223. Sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) are a well-established model to study G-protein modulation of voltage-gated calcium channels (VGCCs). SCG neurons can be easily dissociated and are amendable to heterologous expression of genes, including genetic tools to study G-protein signaling pathways, within a time frame to maintain good spatial voltage-clamp control of membrane potential during electrophysiological recordings (836 h post dissociation). This protocol focuses on examining G-protein modulation of VGCCs; however, the procedures and experimental setup for acute application of agonists can be applied to study modulation of other ion channels (e.g., M-current, G-protein-coupled inwardly rectifying potassium channels). We also discuss some common sources of artifacts that can arise during acute drug application onto dissociated neurons, which can mislead interpretation of results.

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15
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2016
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Alcohol Abuse and Alcoholism
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Lu, Van B; Ikeda, Stephen R (2016) Strategies for Investigating G-Protein Modulation of Voltage-Gated Ca2+ Channels. Cold Spring Harb Protoc 2016:pdb.top087072
Liput, Daniel J; Lu, Van B; Davis, Margaret I et al. (2016) Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia. Sci Rep 6:25137
Lu, Van B; Ikeda, Stephen R (2016) G-Protein Modulation of Voltage-Gated Ca2+ Channels from Isolated Adult Rat Superior Cervical Ganglion Neurons. Cold Spring Harb Protoc 2016:pdb.prot091223
Nguyen, Tuan A; Sarkar, Pabak; Veetil, Jithesh V et al. (2015) Covert Changes in CaMKII Holoenzyme Structure Identified for Activation and Subsequent Interactions. Biophys J 108:2158-70
Puhl Iii, Henry L; Won, Yu-Jin; Lu, Van B et al. (2015) Human GPR42 is a transcribed multisite variant that exhibits copy number polymorphism and is functional when heterologously expressed. Sci Rep 5:12880
Lu, Van B; Ikeda, Stephen R; Puhl 3rd, Henry L (2015) A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal. J Neurosci 35:8021-34
Iyer, Malliga R; Cinar, Resat; Liu, Jie et al. (2015) Structural Basis of Species-Dependent Differential Affinity of 6-Alkoxy-5-Aryl-3-Pyridinecarboxamide Cannabinoid-1 Receptor Antagonists. Mol Pharmacol 88:238-44
Puhl 3rd, Henry L; Lu, Van B; Won, Yu-Jin et al. (2014) Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split. PLoS One 9:e100694
Won, Yu-Jin; Lu, Van B; Puhl 3rd, Henry L et al. (2013) ?-Hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G-protein-coupled receptor FFA3. J Neurosci 33:19314-25
Lu, Van B; Puhl 3rd, Henry L; Ikeda, Stephen R (2013) N-Arachidonyl glycine does not activate G protein-coupled receptor 18 signaling via canonical pathways. Mol Pharmacol 83:267-82

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