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National Institute on Alcohol Abuse and Alcoholism (NIAAA)

LMP - Section on Transmitter Signaling (TS)

Mission Statement:

Focuses on identifying the molecular components of intracellular signaling cascades.

Ion Channel Modulation by Second Messenger Systems

The Section on Transmitter Signaling focuses primarily on determining the molecular mechanisms underlying G-protein coupled receptor (GPCR) modulation of voltage-gated Ca2+ channels in neuronal systems using electrophysiological, optical, molecular, and biochemical techniques. A consequence of modulation, which usually manifests as a decrease in current flow through the channel, neuronal excitability and neurotransmitter release at synapses is modified. Although several signaling pathways have been identified, the best-studied is a direct inhibition of the ion channel by G-protein βγ-subunits liberated from the G-protein heterotrimer following agonist-mediated receptor activation. This canonical pathway is shared among the high-voltage activated Ca2+ channels of the CaV2.x family (CaV2.1–2.3; P/Q-, N-, and R-type, respectively) and represents one of the most widely studied and best understood mechanism of presynaptic inhibition. GPCRs (e.g., CB1, CRF, mGluR, NPY, and nociceptin) comprise major cellular targets for pharmaceuticals used in the treatment of alcoholism and other addictive disorders. Additionally, N-type Ca2+ channels and heterotrimeric G-protein signaling pathways utilizing Gβγ have been directly implicated in ethanol reward and consumption mechanisms.

Optical methods for quantifying protein-protein interactions in living cells
Chen, Puhl

We have previously demonstrated that Förster 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. In this manuscript, we extended 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 H, Puhl HL, Ikeda SR. Estimating protein-protein interaction affinity in living cells using quantitative FRET measurements.
J Biomed Optics 12:054011, 2007.

Calcium channel modulation via atypical cannabinoid-related GPCRs and endogenous ligands
Guo, Williams, Puhl

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 a- 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 GJ3y-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.

In another study, the effects of N-arachidonoyl L-serine (ARA-S), a recently discovered lipoamino acid found in the central nervous system, on N-type calcium channels of rat sympathetic ganglion neurons were 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. ARA-S did not influence G-protein modulation of calcium channels and appeared to act independently of G- protein coupled receptors. These findings provide a foundation for investigating possible roles for ARA-S in nervous system function.


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