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 three main areas of progress during the current funding period. Two of the areas, both related to voltage-gated sodium channels arose from the completion of long standing projects that have been a minor, but significant, focus of the laboratory.? ? The effect of N-arachidonoyl L-serine (ARA-S), a recently discovered lipoamino acid found in the CNS, on N-type calcium channels of rat sympathetic ganglion neurons was determined using whole cell patch clamp. Application of ARA-S produced a rapid and reversible augmentation of calcium current that was voltage dependent and resulted from a hyperpolarizing shift in the activation curve. At depolarized potentials, the same maximal conductance was attained in the presence of ARA-S arguing against a recruitment of covert channels or a change in the maximum probability of opening. Similar results were obtained with the related lipoamino acids N-arachidonoyl L-alanine (ARA-A) and N-arachidonoyl L-glycine (ARA-G). However, application of N-arachidonoyl L-dopamine (ARA-DA) did not produce a significant effect. Further investigation of ARA-S did not reveal an influence on G protein modulation of calcium channels and thus ARA-S actions appeared to occur independently of G-protein-coupled receptors. There were two main implications to these findings. First, the ARA S concentration (10 micromolar) used falls well within the concentration range used to probe potential physiological roles of ARA S and related endocannibinoids. Given the well-established role played by N-type calcium channels in providing calcium for synaptic transmission combined with the nonlinear relationship between intracellular calcium concentration and neurotransmitter, one can easily envision changes in synaptic transmission produced by applying ARA S. Thus, receptor-independent effects will need to be considered before meaningful interpretations of ARA S action are entertained. Second, it is possible that direct actions of ARA S on ion channels underlie physiological processes. Although endocannabinoids and related lipid compounds often have direct effects on ion channel function, the effect of ARA S on N-type calcium channels is somewhat unique. Closely related compounds such as anandamide, 2-arachidonoyl glycerol, and ARA-DA either had no effect or produced inhibition at similar concentrations while both ARA-A and ARA-G were capable of augmenting calcium channel currents to varying degrees. From this series of compounds, the presence of a carboxylic acid group was common to the substances that enhanced ICa amplitude. It seems likely that ARA-S, ARA-G, and ARA-A are negatively charged at pH 7.4 and thus, a possible explanation for the effects of ARA-S and related lipoamino acids is alteration of the membrane surface potential following incorporation of negative charges into the outer leaflet of the plasma membrane. These findings provide a foundation for investigating possible roles for ARA-S in nervous system function. Guo et al., J Neurophysiol. 100:11471151, 2008.? ? Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Na(v)1.8, encoded by the Scn10a gene. Na(v)1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the messenger RNA 5 end, alternative splicing within the 5 UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between -1.6 and -0.2 kilobases of the transcription start site cluster, and a distal sensory neuron switch region beyond -1.6 kilobases that restricted fluorescent protein expression to a subset of primary sensory neurons. The identification of a gene regulatory region from such a tightly regulated gene encoding such a physiologically important gene product makes this characterization unique and could facilitate the identification of nociceptive neurons and perhaps uncover new avenues for the discovery of therapeutic targets for the treatment of chronic pain and multiple sclerosis. We are currently developing transgenic mouse lines, in collaboration with Dr. Rui Costa, to further investigate the nature of the promotor region in vivo. Puhl HL, Ikeda SR. J. Neurochem 106:12091224, 2008.? ? The tetrodotoxin (TTX)-resistant sodium current arising from Na(v)1.8 containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. In this study, we demonstrated that injection of cDNA encoding mouse Na(v)1.8 into the nuclei of rat superior cervical ganglion neurons results in TTX-resistant sodium currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion neurons. The activation and inactivation properties of the heterologously expressed Na(v)1.8 sodium channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed Na(v)1.8 channels were shifted approximately 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of Na(v)1.8 did not substantially affect functional expression in superior cervical ganglion neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged Na(v)1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum thus providing a basis for poor functional expression in clonal cell lines. Our results establish sympathetic neurons as a favorable surrogate for the expression and study of molecularly defined Na(v)1.8 -containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of Na(v)1.8 with a biophysical phenotype identical to that found in sensory neurons. Schofield GG, Puhl HL, Ikeda. J Neurophysiol 99:19171927, 2008.
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