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1.
J Gen Physiol ; 122(4): 419-44, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12975449

ABSTRACT

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.


Subject(s)
Eye Proteins , Photons , Retinal Rod Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/radiation effects , Rhodopsin/chemistry , Adenosine Triphosphate/metabolism , Animals , Biotransformation , Calcium Signaling/physiology , G-Protein-Coupled Receptor Kinase 1 , Guanosine Triphosphate/metabolism , Kinetics , Membrane Potentials/physiology , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , Patch-Clamp Techniques , Phosphorylation , Protein Kinases/genetics , Protein Kinases/physiology , Reproducibility of Results , Signal Transduction/physiology , Vertebrates
2.
Biochemistry ; 40(32): 9647-56, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11583165

ABSTRACT

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, with very high affinity. Yet, it is common to find that substantial amounts (30-90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become "denatured" during formation of the obligatory nucleotide-free or empty (MT) Galpha-state that is intermediary to GDP/GTP exchange at a single binding site. We find Galpha native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Galpha is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucleotide binding at micromolar ambient GTPgammaS concentrations. Because of the weak preequilibrium, quantitative protection against Galpha aggregation is only achieved at free nucleotide concentrations 10-100 times higher than those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an intramolecular Galpha refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Galpha x GTP isomers described here, only the second can bind Mg2+, "locking" GTP in place with a large net rise in GTP binding affinity. A companion Galpha x GDP isomerization reaction is identified as the cause of the very slow spontaneous GDP dissociation that characterizes G protein nucleotide exchange and low spontaneous background activity in the absence of GPCR activation. Galpha x GDP and Galpha x GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.


Subject(s)
Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Magnesium/metabolism , Transducin/chemistry , Animals , Cattle , Circular Dichroism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Models, Theoretical , Protein Binding , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits , Retinal Rod Photoreceptor Cells/chemistry , Spectrometry, Fluorescence , Time Factors , Transducin/metabolism
3.
Biochemistry ; 40(16): 5075-80, 2001 Apr 24.
Article in English | MEDLINE | ID: mdl-11305924

ABSTRACT

Xenon and dichloromethane are inhalational anesthetic agents whose binding to myoglobin has been demonstrated by X-ray crystallography. We explore the thermodynamic significance of such binding using differential scanning calorimetry, circular dichroism spectroscopy, and hydrogen-tritium exchange measurements to study the effect of these agents on myoglobin folding stability. Though specific binding of these anesthetics might be expected to stabilize myoglobin against unfolding, dichloromethane actually destabilized myoglobin at all examined concentrations of this anesthetic (15, 40, and 200 mM). On the other hand, xenon (1 atm) stabilized myoglobin. Thus, dichloromethane and xenon have opposite effects on myoglobin stability despite localization in comparably folded X-ray crystallographic structures. These results suggest a need for solution measurements to complement crystallography if the consequences of weak binding to proteins are to be appreciated.


Subject(s)
Anesthetics, Inhalation/chemistry , Crystallography, X-Ray , Methylene Chloride/chemistry , Myoglobin/chemistry , Animals , Calorimetry, Differential Scanning , Circular Dichroism , Crystallography, X-Ray/methods , Horses , Hydrogen/chemistry , Protein Binding , Protein Folding , Recombinant Proteins/chemistry , Thermodynamics , Tritium/chemistry , Whales , Xenon/chemistry
4.
Proteins ; 42(4): 436-41, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11170198

ABSTRACT

Firefly luciferase is considered a reasonable model of in vivo anesthetic targets despite being destabilized by anesthetics, as reflected by differential scanning calorimetry (DSC). We examined the interaction between two inhaled anesthetics, ATP, luciferase, and temperature, using amide hydrogen exchange, tryptophan fluorescence, and photolabeling in an attempt to examine this apparent discrepancy. In the absence of ATP/Mg2+, halothane and bromoform cause destabilization, as measured by hydrogen exchange, suggesting nonspecific interactions. In the presence of ATP/Mg2+ and at room temperature, the anesthetics produce considerable stabilization with a negative DeltaH, indicating population of a conformer with a specific anesthetic binding site. Stabilizing interactions are lost, however, at unfolding temperatures. We suggest that preferential binding to aggregated forms of luciferase explain the higher temperature destabilization detected with DSC. Our results demonstrate a cooperative binding equilibrium between native ligands and anesthetics, suggesting that similar interactions could underlie actions at biologically relevant targets.


Subject(s)
Adenosine Triphosphate/metabolism , Anesthetics, Inhalation/metabolism , Luciferases/metabolism , Recombinant Proteins/chemistry , Fluorescence , Halothane/metabolism , Hydrogen/chemistry , Luminescent Measurements , Photoaffinity Labels/chemistry , Protein Folding , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Trihalomethanes/metabolism , Tryptophan/chemistry
5.
Biochemistry ; 39(29): 8497-502, 2000 Jul 25.
Article in English | MEDLINE | ID: mdl-10913255

ABSTRACT

General anesthetics have been reported to alter the functions of G protein coupled receptor (GPCR) signaling systems. To determine whether these effects might be mediated by direct binding interactions with the GPCR or its associated G protein, we studied the binding character of halothane on mammalian rhodopsin, structurally the best understood GPCR, by using direct photoaffinity labeling with [(14)C]halothane. In the bleached bovine rod disk membranes (RDM), opsin and membrane lipids were dominantly photolabeled with [(14)C]halothane, but none of the three G protein subunits were labeled. In opsin itself, halothane labeling was inhibited by unlabeled halothane with an IC(50) of 0.9 mM and a Hill coefficient of -0.8. The stoichiometry was 1.1:1.0 (halothane:opsin molar ratio). The IC(50) values of isoflurane and 1-chloro-1,2, 2-trifluorocyclobutane were 5.0 and 15 mM, respectively. Ethanol had no effect on opsin labeling by halothane. A nonimmobilizer, 1, 2-dichlorohexafluorocyclobutane, inhibited halothane labeling by 50% at 0.05 mM. The present results demonstrate that halothane binds specifically and selectively to GPCRs in the RDM. The absence of halothane binding to any of the G protein subunits strongly suggests that the functional effects of halothane on GPCR signaling systems are mediated by direct interactions with receptor proteins.


Subject(s)
Anesthetics, Inhalation/metabolism , GTP-Binding Proteins/metabolism , Halothane/metabolism , Receptors, Cell Surface/metabolism , Retina/metabolism , Affinity Labels , Anesthetics, Inhalation/pharmacology , Animals , Binding Sites , Cattle , Halothane/pharmacology , In Vitro Techniques , Kinetics , Receptors, Cell Surface/drug effects , Signal Transduction/drug effects
6.
Biochemistry ; 39(19): 5738-49, 2000 May 16.
Article in English | MEDLINE | ID: mdl-10801324

ABSTRACT

Reduced effector activity and binding of arrestin are widely accepted consequences of GPCR phosphorylation. However, the effect of receptor multiphosphorylation on G protein activation and arrestin binding parameters has not previously been quantitatively examined. We have found receptor phosphorylation to alter both G protein and arrestin binding constants for light-activated rhodopsin in proportion to phosphorylation stoichiometry. Rod disk membranes containing different average receptor phosphorylation stoichiometries were combined with G protein or arrestin, and titrated with a series of brief light flashes. Binding of G(t) or arrestin to activated rhodopsin augmented the 390 nm MII optical absorption signal by stabilizing MII as MII.G or MII.Arr. The concentration of active arrestin or G(t) and the binding constant of each to MII were determined using a nonlinear least-squares (Simplex) reaction model analysis of the titration data. The binding affinity of phosphorylated MII for G(t) decreased while that for arrestin increased with each added phosphate. G(t) binds more tightly to MII at phosphorylation levels less than or equal to two phosphates per rhodopsin; at higher phosphorylation levels, arrestin binding is favored. However, arrestin was found to bind much more slowly than G(t) at all phosphorylation levels, perhaps allowing time for phosphorylation to gradually reduce receptor-G protein interaction before arrestin capping of rhodopsin. Sensitivity of the binding constants to ionic strength suggests that a strong membrane electrostatic component is involved in both the reduction of G(t) binding and the increase of arrestin binding with increasing rhodopsin phosphorylation.


Subject(s)
Arrestin/metabolism , GTP-Binding Proteins/metabolism , Light , Rhodopsin/metabolism , Algorithms , Animals , Cattle , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Hydrogen-Ion Concentration , Kinetics , Osmolar Concentration , Phosphorylation , Protein Binding , Rhodopsin/analogs & derivatives , Static Electricity
7.
Biochemistry ; 38(34): 11103-14, 1999 Aug 24.
Article in English | MEDLINE | ID: mdl-10460166

ABSTRACT

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.


Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/physiology , Rhodopsin/chemistry , Rhodopsin/metabolism , Animals , Cattle , Hydrogen-Ion Concentration , Kinetics , Membrane Potentials , Membrane Proteins/metabolism , Osmolar Concentration , Phosphorylation , Photolysis , Protons , Rhodopsin/physiology , Rod Cell Outer Segment/physiology , Spectrophotometry , Surface Properties
8.
Biochemistry ; 38(21): 6862-78, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10346908

ABSTRACT

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.


Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Rhodopsin/analogs & derivatives , Rod Cell Outer Segment/chemistry , Rod Cell Outer Segment/metabolism , Animals , Cattle , Hydrogen-Ion Concentration , Kinetics , Light , Mathematical Computing , Monte Carlo Method , Photolysis , Protein Binding , Protein Conformation , Reproducibility of Results , Rhodopsin/chemistry , Rhodopsin/metabolism , Scattering, Radiation , Solutions , Spectrophotometry, Ultraviolet , Temperature , Titrimetry
9.
Biochim Biophys Acta ; 1430(1): 46-56, 1999 Feb 10.
Article in English | MEDLINE | ID: mdl-10082932

ABSTRACT

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.


Subject(s)
Anesthetics, Inhalation/pharmacology , Halothane/pharmacology , Protein Folding , Serum Albumin, Bovine/chemistry , Calorimetry, Differential Scanning , Serum Albumin, Bovine/metabolism
10.
Biochemistry ; 37(33): 11393-8, 1998 Aug 18.
Article in English | MEDLINE | ID: mdl-9708973

ABSTRACT

Deactivation of many G protein coupled receptors (GPCRs) is now known to require phosphorylation of the activated receptor. The first such GPCR so analyzed was rhodopsin, which upon light activation forms an intramolecular equilibrium between the two conformers, metarhodopsin I and II (MI and MII). In this study, we find surprisingly that rhodopsin phosphorylation increases rather than diminishes the formation of MII, the conformation that activates G protein. The MI-MII equilibrium constant was progressively shifted toward MII as the experimental phosphorylation stoichiometry was increased from 0 to 6.4 phosphates per rhodopsin. Increasing phosphorylation both increased MII's formation rate (k1) and decreased its rate of loss (k-1). The direct effect of cytoplasmic surface phosphorylation on intramolecular conformer equilibria observed here may be important to functional state modulation of other membrane proteins.


Subject(s)
Protein Conformation , Rhodopsin/chemistry , Rhodopsin/metabolism , Animals , Cattle , Hydrogen-Ion Concentration , Kinetics , Light , Phosphorylation , Rhodopsin/analogs & derivatives , Rod Cell Outer Segment/chemistry , Rod Cell Outer Segment/metabolism , Scattering, Radiation , Temperature
11.
Toxicol Lett ; 100-101: 387-91, 1998 Nov 23.
Article in English | MEDLINE | ID: mdl-10049169

ABSTRACT

1. We have used differential scanning calorimetry to measure the halothane induced change in stability of five lipid-free proteins in aqueous solution. 2. The temperature at peak heat capacity (Tm) as the sample is heated provides a measure of stability. 3. Addition of halothane increases Tm for bovine and human serum albumin, but decreases Tm for hen egg white lysozyme, bovine pancreatic ribonuclease A, and horse skeletal muscle myoglobin. 4. A shift of Tm in either direction may model the action of inhaled anesthetics on relevant proteins in the central nervous system.


Subject(s)
Anesthetics, Inhalation/chemistry , Proteins/chemistry , Anesthetics, Inhalation/pharmacology , Animals , Calorimetry, Differential Scanning , Cattle , Drug Stability , Halothane/chemistry , Halothane/pharmacology , Humans , Proteins/drug effects , Thermodynamics
12.
Biochemistry ; 36(18): 5363-71, 1997 May 06.
Article in English | MEDLINE | ID: mdl-9154918

ABSTRACT

The effect of Ca2+ binding to parvalbumin was monitored by probes of conformation including absorption, fluorescence, circular dichroism (CD), infrared (IR) spectroscopy and differential scanning calorimetry. These experimental studies were compared with molecular dynamics computations on the structures of the Ca-bound and Ca-free forms of cod parvalbumin. The UV CD spectra show that removal of calcium results in a decrease in the alpha-helical content of the protein. The IR amide I' and III' regions are very much affected by Ca removal and are indicative of significant perturbation of secondary structure. The fluorescence of tryptophan, the IR markers, and UV ellipticity all show changes with temperature, pointing to a lowering of protein stability upon Ca removal. These results are consistent with the structures obtained for both the Ca-bound and Ca-free proteins after 200 ps of solvated molecular dynamics simulations which show a decrease in the secondary structure upon Ca removal.


Subject(s)
Calcium/metabolism , Computer Simulation , Models, Molecular , Parvalbumins/chemistry , Protein Conformation , Calcium/physiology , Calcium-Binding Proteins/chemistry , Calorimetry, Differential Scanning , Circular Dichroism , Crystallography, X-Ray , Molecular Sequence Data , Parvalbumins/metabolism , Spectrometry, Fluorescence , Spectrophotometry , Spectroscopy, Fourier Transform Infrared , Temperature , Thermodynamics
13.
Biochemistry ; 36(9): 2450-8, 1997 Mar 04.
Article in English | MEDLINE | ID: mdl-9054549

ABSTRACT

The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described. The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala. The overall topology was intended to be similar to that found in the Escherichia coli protein ROP. The design strategy included consideration of geometric complementarity of the packing of side chains within the hydrophobic core as well as the use of specific interfacial interactions, both of which were intended to favor the desired ROP-like topology. Additionally, the sequence was designed to destabilize potential alternative structures that might compete with the desired topology. The peptides (RLP-1, RLP-2, and RLP-3) assemble into stable alpha-helical dimers and exhibit the hallmarks of a native protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes. Also, the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence of the CD spectra and confirmed by differential scanning calorimetry (DSC). The values determined by the two methods are in excellent agreement and are in the range of those of naturally occurring proteins of this size. These results suggest that it is now possible to design native-like helical proteins that should serve as templates for the further design of functional proteins.


Subject(s)
Bacterial Proteins/chemistry , Helix-Loop-Helix Motifs , Protein Engineering/methods , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Centrifugation, Density Gradient , Escherichia coli , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Denaturation , Protein Structure, Secondary , Thermodynamics
14.
Biophys J ; 66(1): 80-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8130348

ABSTRACT

A sample of rhodopsin that is exposed to a series of small light flashes of equal intensity is expected to bleach in successively smaller decrements in proportion to the remaining unbleached rhodopsin. The exponential depletion law describing this effect has been used as a rapid, convenient, and intuitive method for determining the fraction of rhodopsin bleached per flash. This method is commonly assumed to be free of error provided the amount bleached is small, so that there is no significant photoregeneration. We show here, however, that if there is any photoregeneration, the bleach fraction calculated in this manner can be in error by a factor of two or more, no matter how little rhodopsin is bleached. This flaw occurs insidiously, without perturbing the expected exponentiality of the bleaching decrements, thereby escaping ready notice. The erroneous bleach values readily propagate as underestimates of metarhodopsin and accompanying G-protein equilibrium and kinetic constants. We derive equations for correcting such errors and illustrate how empirical constants can be obtained from experiments that permit the true fraction bleached to be determined.


Subject(s)
Light , Rhodopsin/chemistry , Animals , Cattle , Cell Membrane/metabolism , Kinetics , Mathematics , Models, Theoretical , Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/metabolism , Rhodopsin/radiation effects , Spectrophotometry , Time Factors
15.
Vis Neurosci ; 10(3): 473-8, 1993.
Article in English | MEDLINE | ID: mdl-8388245

ABSTRACT

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.


Subject(s)
GTP-Binding Proteins/analysis , Interneurons/chemistry , Ion Channels , Rod Cell Outer Segment/chemistry , Amino Acid Sequence , Animals , Antigens/analysis , Arrestin , Cats , Cattle , Cyclic Nucleotide-Gated Cation Channels , Eye Proteins/analysis , Fluorescent Antibody Technique , Macaca mulatta , Molecular Sequence Data , Phosphodiesterase Inhibitors/analysis , Phosphoric Diester Hydrolases/analysis , Rats , Transducin/analysis , Urodela
16.
J Cell Sci ; 103 ( Pt 1): 157-66, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1429903

ABSTRACT

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this nonaxonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes. This fraction remains membrane-associated after extensive low- or high-salt washing, requiring detergents or protein denaturants for release from ROS membranes. Unlike ROS soluble tubulin it associates tightly with liposomes upon detergent solubilization and reconstitution. The ROS membrane-associated tubulin is highly enriched in isolated ROS plasma membrane fractions compared to the total outer segment membrane pool and can be localized to the plasma membrane but not to disks by immunofluorescent staining, suggesting a possible role in the structure or electrophysiology of the rod outer segment plasma membrane.


Subject(s)
Rod Cell Outer Segment/metabolism , Tubulin/metabolism , Animals , Cattle , Cell Membrane/metabolism , Detergents , Immunohistochemistry , Liposomes , Solubility , Tubulin/isolation & purification
17.
J Biol Chem ; 265(31): 18922-7, 1990 Nov 05.
Article in English | MEDLINE | ID: mdl-2121732

ABSTRACT

The role of GDP has heretofore been little studied in the analysis of visual receptor G-protein (G) interactions. Here we use kinetically resolved absorption and light scattering spectroscopy, centrifugation, porous membrane filtration, and enzyme assay to compare the effectiveness of GDP with that of GTP or gamma-thio-guanosine-5'-triphosphate in the modulation of G-protein binding to rod disc membranes and activated receptor (R*). We also compare effectiveness of GDP with that of GTP in the separation of G alpha and G beta gamma subunits and in activation of effector, cGMP phosphodiesterase. We find that when different nucleotide affinities are taken into account, actions such as the release of G from R* binding, earlier ascribed to GTP alone, are also typical of GDP. The principal specific actions of GTP that occur only weakly or undetectably for GDP are, respectively, the release of G-protein subunits from the membrane into solution and activation of phosphodiesterase. While GDP, like GTP, releases G-protein binding to receptor, we argue that GDP cannot mediate G-protein subunit separation, even on the membrane surface. GDP retained on G-protein after GTP hydrolysis may function to prevent tight binding to quiescent receptors in a manner analogous to its action on G-protein binding to activated receptors. Weak binding of G.GDP may function to accelerate receptor catalyzed amplification during transduction.


Subject(s)
GTP-Binding Proteins/metabolism , Guanosine Diphosphate/pharmacology , Rod Cell Outer Segment/metabolism , Animals , Cattle , Darkness , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/pharmacology , Kinetics , Light , Mathematics , Models, Theoretical , Scattering, Radiation , Spectrophotometry
18.
Nature ; 326(6113): 600-3, 1987.
Article in English | MEDLINE | ID: mdl-2436056

ABSTRACT

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.


Subject(s)
Cyclic GMP/physiology , Ion Channels/physiology , Photoreceptor Cells/physiology , Rod Cell Outer Segment/physiology , Animals , Binding Sites , Calcium/metabolism , Cattle , Cell Membrane/analysis , Proteins/analysis , Rod Cell Outer Segment/analysis
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