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LMP - Section on Transmitter Signaling (TS)
Steven R. Ikeda, MD, PhD Chief
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health
5625 Fishers Lane, Room TS-11A:MSC 9411
Bethesda MD 20892-9413
telephone: +1 301.443.2807
fax: +1 301.480.0466
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
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.
Guo J, Williams, DJ, Puhl HL, Ikeda SR. Activation of GPR35, an orphan G protein coupled receptor, inhibits N-type Ca2+ channels in rat mammalian neurons.
J Pharmacol Exp Ther 324:342–51, 2008.
Guo J, Williams, DJ, Ikeda SR. N-Arachidonyl L-serine, a putative endocannabinoid, alters the activation of N-type Ca2+ channels in sympathetic neurons.
J Neurophysiol 100:1147–51, 2008.
Sensory neuron-specific Na+ channel (NaV 1.8) function and expression
Puhl; in collaboration with Schofield
The tetrodotoxin (TTX)-resistant Na+ current arising from NaV 1.8 containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. In this study, we showed that injection of cDNA encoding mouse NaV1 .8 into the nuclei of rat superior cervical ganglion (SCG) neurons resulted in TTX-resistant Na+ currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed NaV 1.8 Na+ channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed NaV 1.8 channels were shifted approximately 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of NaV 1.8 did not substantially affect functional expression in SCG 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 NaV1.8 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 SCG neurons as a favorable surrogate for the expression and study of molecularly defined NaV 1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of NaV1 .8 with a biophysical phenotype identical to that found in sensory neurons.
In addition, we identified the promoter for the tetrodotoxin insensitive sodium channel, NaV 1.8, which is encoded by the Scn10a gene. NaV1.8 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 mRNA 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 -0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond -1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons.
Schofield GG, Puhl HL, Ikeda SR. Properties of wild-type and fluorescent protein-tagged mouse tetrodotoxin-resistant sodium channel (NaV 1.8) heterologously expressed in rat sympathetic neurons.
J Neurophysiol 99:1917–27, 2008.
Puhl HL, Ikeda SR. Identification of the sensory neuron specific regulatory region for the mouse gene encoding the voltage-gated sodium channel NaV1.8.
J Neurochem 106:1209–24, 2008.
Heterotrimeric G-protein interaction with GoLoco motif containing proteins.
Guo; in collaboration Willard, Zheng, Digby, Kimple, Johnston, Bosch, Willard, Lambert, Du, Siderovski
Heterotrimeric G-protein Got subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Goti) of Got subunits, and thus it is assumed that a Goti/GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Goti subunits that renders a selective loss-of-function for GoLoco motif binding: namely, an asparagine-to¬isoleucine substitution in the otD-otE loop of the Got helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Goti1 association with all human GoLoco motif proteins and abrogated interaction between the C. elegans Got subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of G i subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with GJ31 dimers, adenylyl cyclase and seven transmembrane¬domain receptors. GoLoco-insensitivity rendered Goti subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Goti subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Goti/GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.
Willard FS, Zheng Z, Guo J, Digby GJ, Kimple AJ, Johnston CA, Bosch D, Willard MD, Lambert NA, Ikeda SR, Du Q, Siderovski DP. A point mutation to Goti selectively blocks GoLoco motif binding: Direct evidence for Got/GoLoco complexes in mitotic spindle dynamics.
J Biol Chem 283:36698–7 10, 2008.