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:? ? 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. 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 receptor 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. Guo et al., J Physiol. 576:447492, 2006.? ? GPR35 is a G protein coupled receptor recently de-orphanized using high throughput intracellular calcium measurements in clonal cell lines expressing a chimeric G-protein α-subunit. From these screens, kynurenic acid, an endogenous metabolite of tryptophan, and zaprinast, a synthetic inhibitor of cyclic guanosine monophosphate specific phosphodiesterase, emerged as potential agonists for GPR35. To investigate the coupling of GPR35 to natively expressed neuronal signaling pathways and effectors, we heterologously expressed GPR35 in rat sympathetic neurons and examined the modulation of N-type (CaV2.2) calcium channels. In neurons expressing GPR35, calcium channels were inhibited in the absence of overt agonist indicating a tonic receptor activity. Application of kynurenic acid or zaprinast resulted in robust voltage-dependent calcium current inhibition characteristic of Gβγ-mediated modulation. Both agonist independent and dependent effects of GPR35 were blocked by Bordetella pertussis toxin pretreatment indicating the involvement of Gi/o proteins. In neurons expressing GPR35a, a short splice variant of GPR35, zaprinast was more potent (EC50 = 1 μM) than kynurenic acid (58 μM), but had a similar efficacy (approximately 60% maximal calcium current inhibition). Expression of GPR35b, which has an additional 31 residues at the N-terminus, produced similar results but with much greater variability. Both GPR35a and GPR35b appeared to have similar expression patterns when fused to fluorescent proteins. These results suggest a potential role for GPR35 in regulating neuronal excitability and synaptic release. Guo et al, J. Pharmacol. Exp. Ther., in press, 2007.? ? Finally, we previously demonstrated that Forster resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores can be determined in living cells using 3-cube wide-field fluorescence microscopy (Chen et al, Biophysics J., 91:L3941, 2006) . Here, we extend the methodology to estimate the effective equilibrium dissociation constant (Kd) and the intrinsic FRET efficiency (Emax) of an interacting donor-acceptor pair. Assuming bimolecular interaction, the predicted FRET efficiency is a function of donor concentration, acceptor concentration, Kd, and Emax. We estimated Kd and Emax by minimizing the sum of the squared error (SSE) between the predicted and measured FRET efficiency. This was accomplished by examining the topology of SSE values for a matrix of hypothetical Kd and Emax values. Applying an F-test, the 95% confidence contour of Kd and Emax was calculated. We tested the method by expressing an inducible FRET fusion pair consisting of FKBP12-Cerulean and Frb-Venus in HeLa cells. As the Kd for FKBP12-rapamycin and Frb has been analytically determined, the relative Kd (in fluorescence units) could be calibrated with a value based on protein concentration. The described methodology should be useful for comparing protein-protein interaction affinities in living cells. Chen et al., J. Biomed. Optics., in press, 2007.
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