99 research outputs found
Kinetics of Rhodopsin Deactivation and Its Role in Regulating Recovery and Reproducibility of Rod Photoresponse
The single photon response (SPR) in vertebrate phototransduction is regulated by the dynamics of R* during its lifetime, including the random number of phosphorylations, the catalytic activity and the random sojourn time at each phosphorylation level. Because of this randomness the electrical responses are expected to be inherently variable. However the SPR is highly reproducible. The mechanisms that confer to the SPR such a low variability are not completely understood. The kinetics of rhodopsin deactivation is investigated by a Continuous Time Markov Chain (CTMC) based on the biochemistry of rhodopsin activation and deactivation, interfaced with a spatio-temporal model of phototransduction. The model parameters are extracted from the photoresponse data of both wild type and mutant mice, having variable numbers of phosphorylation sites and, with the same set of parameters, the model reproduces both WT and mutant responses. The sources of variability are dissected into its components, by asking whether a random number of turnoff steps, a random sojourn time between steps, or both, give rise to the known variability. The model shows that only the randomness of the sojourn times in each of the phosphorylated states contributes to the Coefficient of Variation (CV) of the response, whereas the randomness of the number of R* turnoff steps has a negligible effect. These results counter the view that the larger the number of decay steps of R*, the more stable the photoresponse is. Our results indicate that R* shutoff is responsible for the variability of the photoresponse, while the diffusion of the second messengers acts as a variability suppressor
Differential Localization of G Protein βγ Subunits
G protein βγ subunits
play essential roles in regulating
cellular signaling cascades, yet little is known about their distribution
in tissues or their subcellular localization. While previous studies
have suggested specific isoforms may exhibit a wide range of distributions
throughout the central nervous system, a thorough investigation of
the expression patterns of both Gβ and Gγ isoforms within
subcellular fractions has not been conducted. To address this, we
applied a targeted proteomics approach known as multiple-reaction
monitoring to analyze localization patterns of Gβ and Gγ
isoforms in pre- and postsynaptic fractions isolated from cortex,
cerebellum, hippocampus, and striatum. Particular Gβ and Gγ
subunits were found to exhibit distinct regional and subcellular localization
patterns throughout the brain. Significant differences in subcellular
localization between pre- and postsynaptic fractions were observed
within the striatum for most Gβ and Gγ isoforms, while
others exhibited completely unique expression patterns in all four
brain regions examined. Such differences are a prerequisite for understanding
roles of individual subunits in regulating specific signaling pathways
throughout the central nervous system
Closely Related G-Protein-Coupled Receptors Use Multiple And Distinct Domains On G-Protein Α-Subunits For Selective Coupling
The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric Galpha subunits, comprised of various regions of Gi1alpha, Gtalpha, and Gqalpha, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between Gi1alpha and Gtalpha sequences, the 5-HT1A and 5-HT1B serotonin and M2 muscarinic receptors can couple with Gi1 but not Gt. It is possible to distinguish domains that eliminate coupling and are defined as critical, from those that impair coupling and are defined as important. Domains within the N terminus, alpha4-helix, and alpha4-helix-alpha4/beta6-loop of Gi1alpha are involved in 5-HT and M2 receptor interactions. Chimeric Gi1alpha/Gqalpha subunits verify the critical role of the Galpha C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC50 for interactions with Gi1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration
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Rhodopsin Expression Level Affects Rod Outer Segment Morphology and Photoresponse Kinetics
Background: The retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly. Methodology/Principal Findings: The structure and function of rods from hemizygous rhodopsin knockout (R+/−) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/− rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics. Conclusions/Significance: Rhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/− rods to light
Collybolide Is a Novel Biased Agonist of κ-Opioid Receptors With Potent Antipruritic Activity
Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum. Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non–histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects
Antagonists of the Receptor-G Protein Interface Block Gi-coupled Signal Transduction
The carboxyl terminus of heterotrimeric G protein alpha subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Galphai1/2 or Galphao1 impair agonist binding to A1 adenosine receptors, whereas Galphas or Galphat peptides have no effect. Previously, by using a combinatorial library we identified a series of Galphat peptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). Native Galphai1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the Ki, suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Galphai proteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or gamma-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Galphai1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted
Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in Gα i Subunits †, ‡
Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4− reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release
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