Biomedical subjects

Theodore G Wensel

Publications and source records attributed to Theodore G Wensel.

At least 19 recordsLinked to original sources

PubMed2006

Defective development of photoreceptor membranes in a mouse model of recessive retinal degeneration.

Retinal neurodegeneration occurs in several inherited diseases. Some of the most severe disease alleles involve mutations at the C-terminus of rhodopsin, but in no case is the pathogenic mechanism leading to cell death well understood. We have examined a mouse model of recessive retinal degeneration caused by a knock-in of a human rhodopsin-EGFP fusion gene (hrhoG/hrhoG) at the rhodopsin locus. Whereas heterozygous mutant mice were indistinguishable from control mice, homozygous mutant mice had retinal degeneration. We hypothesized that degeneration might be due to aberrant rhodopsin signaling; however, inhibiting signaling by rearing mice in total darkness had no effect on the rate of degeneration. Using confocal and electron microscopy, we identified the fundamental defect as failed biogenesis of disk membranes, which is observed at the earliest stages of outer segment development. These results reveal that in addition to its role in transport and sorting of rhodopsin to disk membranes, rhodopsin is also essential for formation of disks.

Animals↗

PubMed2006

RGS expression rate-limits recovery of rod photoresponses.

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.

Animals↗

PubMed2006

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer.

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.

Binding Sites↗

PubMed2006

Electrostatic steering at acetylcholine binding sites.

The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.

Acetylcholine↗

PubMed2006

Clearance of intravitreal moxifloxacin.

PURPOSE: To study the clearance of moxifloxacin after intravitreal injection in rabbits. METHODS: Intravitreal injections of 200 microg/0.1 mL of moxifloxacin were administered to rabbits. Four eyes per time point after injection (1 hour and 6, 12, 24, and 36 hours) and three eyes at 48 hours, respectively, were enucleated and immediately frozen and stored at -80 degrees C. Ocular dissection and isolation of frozen vitreous was performed. Vitreous samples were acquired at the various time intervals after injection. Antibiotic assays were performed with high performance liquid chromatography. RESULTS: The concentration of intravitreal moxifloxacin showed an exponential decay with a half-life of 1.72 hours. The mean vitreous concentration was 120.49 +/- 49.23 microg/mL 1 hour after injection, and declined to 20.23 +/- 5.85 microg/mL at 6 hours and 1.06 +/- 0.81 microg/mL at 12 hours, respectively. CONCLUSIONS: The vitreous concentrations achieved were several orders of magnitude greater than the MIC90 of organisms commonly involved in bacterial endophthalmitis, and therapeutic levels were maintained at 12 hours in uninflamed, phakic rabbit eyes. The pharmacokinetic data suggest that intravitreal moxifloxacin may have a role in the treatment of bacterial endophthalmitis.

Animals↗

PubMed2005

Rhodopsin-EGFP knock-ins for imaging quantal gene alterations.

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

Animals↗

PubMed2005

ABCA4 mutations causing mislocalization are found frequently in patients with severe retinal dystrophies.

ABCA4, also called ABCR, is a retinal-specific member of the ATP-binding cassette (ABC) family that functions in photoreceptor outer segments as a flipase of all-trans retinal. Homozygous and compound heterozygous ABCA4 mutations are associated with various autosomal recessive retinal dystrophies, whereas heterozygous ABCA4 mutations have been associated with dominant susceptibility to age-related macular degeneration in both humans and mice. We analyzed a cohort of 29 arRP families for the mutations in ABCA4 with a commercial microarray, ABCR-400 in addition to direct sequencing and segregation analysis, and identified both mutant alleles in two families (7%): compound heterozygosity for missense (R602W) and nonsense (R408X) alleles and homozygosity for a complex [L541P; A1038V] allele. The missense mutations were analyzed functionally in the photoreceptors of Xenopus laevis tadpoles, which revealed mislocalization of ABCA4 protein. These mutations cause retention of ABCA4 in the photoreceptor inner segment, likely by impairing correct folding, resulting in the total absence of physiologic protein function. Patients with different retinal dystrophies harboring two misfolding alleles exhibit early age-of-onset (AO) (5-12 years) of retinal disease. Our data suggest that a class of ABCA4 mutants may be an important determinant of the AO of disease.

ATP-Binding Cassette Transporters↗

PubMed2005

Purification, reconstitution on lipid vesicles, and assays of PDE6 and its activator G protein, transducin.

PDE6 in rod and cone photoreceptors is the principal effector of phototransduction. It is kept at a very low activity level in the dark, and in the light it is strongly activated by the guanosine 5'-triphosphate-bound form of the alpha-subunit of the G protein, transducin. Both transducin and PDE6 are peripheral proteins, and understanding both their interactions with one another and the roles of lipids in their function requires reconstituting purified proteins on the surfaces of defined lipid bilayers. We describe here methods for purifying the proteins, reconstituting them with vesicles, and assaying catalytic activity and binding.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2005

Intravitreal clearance of moxifloxacin.

PURPOSE: To study the clearance of a single dose of intravitreally injected moxifloxacin in rabbits. METHODS: Intravitreal injections of 200 microg/0.1 mL of moxifloxacin were performed in rabbits. Four eyes per time interval after injection (1, 6, 12, 24, 36 hours) and three eyes at 48 hours were enucleated, immediately frozen, and placed at -80 degrees C. Ocular dissection and isolation of frozen vitreous were performed. Antibiotic assays were performed with use of high-performance liquid chromatography. RESULTS: The concentration of intravitreal moxifloxacin showed an exponential decay with a half-life of 1.72 hours. The mean vitreous concentration was 120.49 +/- 49.23 microg/mL 1 hour after injection, which declined to 20.23 +/- 5.85 microg/mL at 6 hours and 1.06 +/- 0.81 microg/mL at 12 hours. The aqueous levels of moxifloxacin showed an exponential decay from 10 microg/mL at 1 hour after intravitreal injection to undetectable levels by 12 hours after injection. CONCLUSIONS: Moxifloxacin clearance from the vitreous is rapid and consistent with previous clearance studies of ciprofloxacin. Given that the injected dose corresponds to several times the minimum inhibitory concentration at which 90% of isolates are inhibited (MIC90) of organisms commonly involved in endophthalmitis, and that therapeutic levels are present up to 12 hours after injection, intravitreal moxifloxacin may have a role in the treatment of endophthalmitis.

Animals↗

PubMed2004

Knock-in human rhodopsin-GFP fusions as mouse models for human disease and targets for gene therapy.

The human rhodopsin gene is the locus for numerous alleles linked to the neurodegenerative disease retinitis pigmentosa. To facilitate the study of retinal degeneration and to test reagents designed to alter the structure and function of this gene, we have developed strains of mice whose native rhodopsin gene has been replaced with the corresponding human DNA modified to encode an enhanced GFP fusion at the C terminus of rhodopsin. The human rhodopsin-GFP fusion faithfully mimics the expression and distribution of wild-type rhodopsin in heterozygotes and serves as a sensitive reporter of rod-cell structure and integrity. In homozygotes, however, the gene induces progressive retinal degeneration bearing many of the hallmarks of recessive retinitis pigmentosa. When the gene is flanked by recognition sites for Cre recombinase, protein expression is reduced approximately 5-fold despite undiminished mRNA levels, suggesting translation inhibition. GFP-tagged human rhodopsin provides a sensitive method to monitor the development of normal and diseased retinas in dissected samples, and it offers a noninvasive means to observe the progress of retinal degeneration and the efficacy of gene-based therapies in whole animals.

Animals↗

PubMed2004

How a G protein binds a membrane.

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.

Animals↗

PubMed2004

Characterization of R9AP, a membrane anchor for the photoreceptor GTPase-accelerating protein, RGS9-1.

The proper recovery of photoreceptor light responses requires timely inactivation of the G-protein transducin (Gt) by GTP hydrolysis. It is now well established that the GTPase-accelerating protein (GAP) RGS9-1 plays an important role in determining the recovery kinetics of photoresponses. RGS9-1 has been found to be anchored to photoreceptor disk membranes by a novel photoreceptor protein, R9AP. R9AP has a single transmembrane domain at its C-terminal region. Membrane tethering by R9AP enhances RGS9-1 GAP activity in vitro and has been hypothesized to be important for the regulation of RGS9-1 function in vivo. In addition, R9AP shows structural similarity to the SNARE complex protein syntaxin and has been shown to be required for the correct targeting and localization of the RGS9-1 protein in photoreceptors. Therefore, R9AP may have additional functions other than that in the phototransduction pathway. This article presents methods and protocols developed for the functional characterization of R9AP in phototransduction, including the immunoprecipitation of the endogenous protein, the expression and purification of recombinant proteins, the reconstitution of proteoliposomes, and assays for its interaction with RGS9-1 and its effects on RGS9-1 GAP activity. These methods may also be applied to the study of R9AP function in other pathways or other cell types or to the studies of other membrane proteins that are structurally similar to R9AP.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Enhancement of phototransduction g protein-effector interactions by phosphoinositides.

Light responses in photoreceptor cells are mediated by the action of the G protein transducin (G(t)) on the effector enzyme cGMP phosphodiesterase (PDE6) at the surface of disk membranes. The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but it has remained uncertain what role phosphoinositides play in vertebrate phototransduction. Reconstitution of PDE6 and activated G(alphat), on the surface of large unilamellar vesicles containing d-myo-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), stimulated PDE activity nearly 4-fold above the level observed with membranes containing no phosphoinositides, whereas G protein-independent activation by trypsin was unaffected by the presence of phosphoinositides. PDE activity was similarly stimulated by d-myo-phosphatidylinositol-3,4-bisphosphate and d-myo-phosphatidylinositol-4-phosphate (PI(4)P), but much less by d-myo-phosphatidylinositol-5-phosphate (PI(5)P) or d-myo-phosphatidylinositol-3,5-bisphosphate. Incubation of rod outer segment membranes with phosphoinositide-specific phospholipase C decreased G protein-stimulated activation of endogenous PDE6, but not trypsin-stimulated PDE activity. Binding experiments using phosphoinositide-containing vesicles revealed patterns of PDE6 binding and PDE6-enhanced G(alphat)-GTPgammaS binding, consistent with the activation profile PI(4,5)P(2) > PI(4)P > PI(5)P approximately control vesicles. These results suggest that enhancement of effector-G protein interactions represents a possible mechanism for modulation of phototransduction gain by changes in phosphoinositide levels, perhaps occurring in response to longterm changes in illumination or other environmental cues.

Animals↗

PubMed2003

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions.

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.

Amino Acid Sequence↗

PubMed2003

From molecules to behavior: new clues for RGS function in the striatum.

RGS proteins act as negative regulators of G protein signaling, and there is growing evidence that the RGS family is important for regulating signaling in neurons. Two articles in this issue of Neuron (Martemyanov et al. and Rahman et al.) shed light on the function of one family member, RGS9-2, in behavioral responses to dopamine signaling in the striatum and on the relationship between its structure and its function.

Animals↗

PubMed2003

Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5.

RGS (regulator of G protein signaling) proteins containing the G protein gamma-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein beta-subunit family, Gbeta5. This interaction is necessary for the stability of both the RGS protein and for Gbeta5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gbeta5 levels may be limiting. To examine further the interactions of Gbeta5 and the GGL domain-containing RGS proteins, we inactivated the Gbeta5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Ggamma2 and RGS4 proteins remain normal in the absence of Gbeta5. The homozygous Gbeta5 knockout (Gbeta5-/-) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gbeta5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gbeta5 and all four RGS proteins that form protein complexes with Gbeta5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.

Animals↗

PubMed2003

GTPase regulators and photoresponses in cones of the eastern chipmunk.

Vertebrate cone and rod photoreceptor cells use similar mechanisms to transduce light signals into electrical signals, but their responses to light differ in sensitivity and kinetics. To assess the role of G-protein GTP hydrolysis kinetics in mammalian cone photoresponses, we have characterized photoresponses and GTPase regulatory components of cones and rods from the cone-dominant retina of the eastern chipmunk. Sensitivity, based on the stimulus strength required for a half-maximum response, of the M-cone population was 38-fold lower than that of the rods. The relatively lower cone sensitivity could be attributed in part to lower amplification in the rising phase and in part to faster recovery kinetics. At a molecular level, cloning of chipmunk cDNA and expression of recombinant proteins provided standards for quantitative immunoblot analysis of proteins involved in GTPase acceleration. The ratio of the cGMP-phosphodiesterase inhibitory subunit gamma to cone pigment, 1:68, was similar to the levels observed for ratios to rhodopsin in bovine retina, 1:76, or mouse retina, 1:65. In contrast, the ratio to pigment of the GTPase-accelerating protein RGS9-1 was 1:62, more than 10 times higher than ratios observed in rod-dominant retinas. Immunoprecipitation experiments revealed that, in contrast to rods, RGS9-1 in chipmunk retina is associated with both the short and long isoforms of its partner subunit G(beta5). The much higher levels of the GTPase-accelerating protein complex in cones, compared with rods, suggest a role for GTPase acceleration in obtaining rapid photoresponse kinetics.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2003

Activation of RGS9-1GTPase acceleration by its membrane anchor, R9AP.

The GTPase-accelerating protein (GAP) complex RGS9-1.G beta(5) plays an important role in the kinetics of light responses by accelerating the GTP hydrolysis of G alpha(t) in vertebrate photoreceptors. Much, but not all, of this complex is tethered to disk membranes by the transmembrane protein R9AP. To determine the effect of the R9AP membrane complex on GAP activity, we purified recombinant R9AP and reconstituted it into lipid vesicles along with the photon receptor rhodopsin. Full-length RGS9-1.G beta(5) bound to R9AP-containing vesicles with high affinity (K(d) < 10 nm), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound with much lower affinity, and binding of those lacking the entire N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin domain plus intervening domain) was not detectable. Formation of the membrane-bound complex with R9AP increased RGS9-1 GAP activity by a factor of 4. Vesicle titrations revealed that on the time scale of phototransduction, the entire reaction sequence from GTP uptake to GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange of G alpha(t) between membrane surfaces is much slower than hydrolysis. Because in rod cells different pools exist of RGS9-1.G beta(5) that are either associated with R9AP or not, regulation of the association between R9AP and RGS9-1.G beta(5) represents a potential mechanism for the regulation of recovery kinetics.

Adaptor Proteins, Signal Transducing↗