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. A project, completed in collaboration with Dr. Fumihito Ono, resulted in the development of a zebrafish neuronal system suitable for investigating sodium and calcium channel modulation and function during a development stage that leverages the genetic malleability and optical transparency of this model organism. The published findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies utilizing this model system. Won YJ, Ono F, Ikeda SR. Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons. PLoS One. 2012;7(8):e42602. A second study was completed that examined a putative endogenous ligand for the G-protein coupled receptor GPR18. Recent studies proposed that N-arachidonyl glycine (NAGly), a carboxylic analog of anandamide, was an endogenous ligand of GPR18. However, other studies failed to detect activation of GPR18 by NAGly. To address this inconsistency, we investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. We heterologously expressed GPR18 in rat sympathetic neurons and examined the modulation of N-type calcium channels. Proper expression and trafficking of receptor was confirmed by the rim-like fluorescence of fluorescently-tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents, but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported by our laboratory (Guo et al., 2008;J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors, G protein-coupled inwardly-rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple pathway utilizing to Gs, Gz, or G15. These results argue that NAGly is not an agonist for GPR18 or that GPR18 signaling involves non-canonical pathways not examined in these studies. Lu, VB, Puhl, HL, Ikeda, SR. (2013) N-arachidonyl glycine (NAGly does not activate G protein-coupled receptor 18 (GPR18) signaling via canonical pathways. Mol Pharmacol 83:267-82. A third project involves the investigation of a recently """"""""de-orphanized"""""""" G-protein coupled receptor termed GPR41 or FFAR3 in rodent sympathetic neurons. These receptors use short-chained fatty acids (SCFA;e.g., acetate and propionate) as endogenous ligands but little else is known. We have discovered that GPR41 is natively expressed and functionally coupled to calcium channels in sympathetic neurons (primarily the celiac/superior mesenteric ganglia). We have also generated evidence that receptor expression is highest (and possibly functional confined to) paravertebral and prevertebral sympathetic neurons. Finally, our data indicate that a ketone body, beta-hydroxybutyrate, acts as an agonist at this receptor. These findings are potentially important for several reasons. First, GPR41 may be expressed in neurons that innervate adipose tissue and thus be an important target for anti-obesity drugs. Second, we proposed that beta-hydroxybutyrate may serve as the primary endogenous ligand for sympathetic ganglia. This finding has implications for conditions in which plasma beta hydroxybutyrate is elevated such as diabetic ketoacidosis or ketogenic diets. Third, acetate is a major metabolite of ethanol. Hence, GPR41 may be involved in both the response to ethanol (especially hangover symptoms) and addiction. It is possible that polymorphisms in GPR41 and primate-specific segmental duplication GPR42 contribute to susceptibility to alcoholism in humans. Fourth, as GPR41 is likely capable of detecting ethanol indirectly via blood acetate levels, it may be possible to leverage this property to interrupt neural pathways contributing to addiction in model organisms using genetic techniques. Won Y-J, Lu, VB, Puhl HL, Ikeda SR. (2013) Beta-hydroxybutyrate modulates N-type calcium channels in rat sympathetic neurons by acting as an agonist for the G protein coupled receptor FFA3 (GPR41). In revision.

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National Institute on 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
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
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
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
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
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) β-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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