Klaus Peter Hofmann

Complex formation between metarhodopsin II and GTP-binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium

1. INTRODUCTION The transition of metarhodopsin I (MI) to meta- rhodopsin II (MII) is the last step in the decay reaction chain of vertebrate rhodopsin photopro- ducts which occurs on a ms time scale and which is therefore sufficiently rapid to be involved in trig- gering visual transduction [I]. The two photo- products MI and MI1 are in a temperature- and pH-dependent equilibrium while MI1 slowly decays further. This equilibrium has been extensively stud- ied (reviewed in 121). We have shown that a first flash bleaching 2% rhodopsin in dark-adapted rod outer segment (ROS) membranes does not lead to the ‘normally’ observed equilibrium of MI and MII; in contrast, MI1 is virtually the only photo- product formed [2]. With increasing photolysis by further flashes delivered on the same sample, more and more MI per flash is formed. Only at bleaching levels 1 lo%, further flashes produce the ‘normal’ mixture of MI and MI1 described by the classical MI/MI1 equilibrium [2]. light-scattering ‘binding signal’ is related to the stoichiometric association of GTP-binding protein (G-protein) to photoexcited rhodopsin [5]. The for- mation of ‘extra MII’ is determined by some peri- pheral protein factor of the disc membrane (21. This study provides evidence that the formation of ‘extra MII’ is caused by a shift of the MI/II equilibrium due to rapid binding of the G-protein to MII. 2. MATERIALS AND METHODS All spectroscopic measurements were done in isotonic saline containing 130 mM KCl, 0.5 mM MgCl2, 1 mM CaC12, 0.5 mM ethylene-diamine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 10 mM piperazine- 1,4-diethane sulfonic acid (PIPES) (pH 7.5). The bleaching range (0- 10%) in which this ‘anom- alously’ high ratio of MI1 vs MI is formed (‘extra MII’-formation) is the same range in which also light-evoked, rapid changes of the scattered light intensity, the so-called ‘P-signals’ [3,4] or ‘binding signal’ [5], are observed, suggesting that these effects may be closely related to one another. The

Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods

A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively.

Measurements of fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells

AbstractFlash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles.We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin.The following results are discussed:1.The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin).2.At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course:N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane.nNi: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect.nP: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. “P” may reflect an influence of the light-induced transmitter release on disc shape and/or mass.

Binding of inositol phosphates to arrestin

Arrestin binds to phosphorylated rhodopsin in its light‐activated form (metarhodopsin II), blocking thereby its interaction with the G‐protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin‐rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5‐tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 μM and 5 μM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.

Shift in the relation between flash-induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes

In the light-induced reaction chain of vertebrate rhodopsin, the transition from metarhodopsin I (M I)’ to metarhodopsin II (M II) is the last reaction which is fast enough to be involved in visual transduction. This reaction, on the other hand, is the first which is drastically influenced by intermolecular interactions of the rhodopsin molecule. The reaction only takes place in the presence of water [ 11, rhodopsin becomes protonated [2] and detergents have a pronounced effect on the reaction kinetics [3]. After thorough bleaching, M II is in a pHand temperature-dependent equilibrium with M I [4]; equal amounts of M I and M II are found at pH 7, T = 3°C. High temperature and low pH favour M II. A similar pHand temperature-dependent equilibrium is also found in rod outer segments (ROS) [ 51. The equilibrium between the species is also dependent on the outer pressure [6] and the membrane surface potential [7]. It can be shifted by pressure jumps [8] and by T-jumps [9]. The kinetics of the shift is then comparable to the light-induced rise of M II. . The equilibrium can be taken as a further monitor Evidence will be provided that the ‘normal’ dependence of the equilibrium on pH and temperature is only found for bleachings >lO%.

Measurement of fast light-induced disc shrinkage within bovine rod outer segments by means of a light-scattering transient

Abstract A fast light-induced light-scattering transient, previously found in rod outer segment suspension, the so-called P-signal (Hofmann, K.P., Uhl, R., Hoffmann, W. and Kreutz, W. (1976) Biophys. Struct. Mechanism 2, 61–77), is described in more detail. The effect has the same action spectrum as rhodopsin bleaching. It is not regenerated with 11-cis retinal. The response is not linear with light-intensity for flashes which bleach more than 2.0% of rhodopsin; it saturates at an intensity corresponding to 15% rhodopsin bleaching. The wavelength- and scattering angle dependence lead to the conclusion that the change in light-scattering reflects a shrinkage of an osmotic compartment of the rod outer segment. The only compartment which we found to be intact in our rod outer segment preparations was the disc or rod sac; therefore, the effect must be attributed to a light-induced shrinkage of the rhodopsin-containing disc organelles. The overall effect (15% of rhodopsin is bleached) is in the range of 0.5–1.5% of the original volume. A light-induced passive cation-efflux from the disc, e.g. of Ca 2+ , can be ruled out as a possible molecular origin of the disc-shrinkage in our preparations.

Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein.

SummaryFormation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (RM) leads to a change in the light scattering of the disk membranes (binding signal or signalP). The signal measured on isolated disks (so-calledPD signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than theRMG binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed thePD-signal level and kinetics as a function of flash intensity and compared it to theRMG-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of thePD signals decrease with flash intensity when the signals are normalized to the same final level. This finding prevents an explanation of the scattering signal by a slow postponed reaction of theRMG complex. We propose to interpret the scattering change as a redistribution of G-protein between a membrane-bound and a solved state. The process is driven by the complexation of membrane-bound G to flash-activated rhodopsin (RM). The experimental evidence for this two-state model is the following: (1) The intensity dependence of the initial rate of thePD signal is explained by the model. Under the assumption of a bimolecular reaction of free G with sites at the membrane, equal to rhodopsin in their concentration, the measured rates yield aKD of 10−5M. (2) Evaluation of the extra MII kinetics yields a biphasic rise at saturating flashes. The measured rates fit to the supply of free and membrane-bound G-protein for the reaction withRM. (3) Quantitative estimation of the expected scattering intensity changes gives a comprehensive description of binding signal and dissociation signal by the gain and loss of G-protein scattering mass. (4) The temperature dependence of thePD-signal rate leads to an activation energy of the membrane-association process ofEa=44 kJ/mol.

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments.

The kinetics of the metarhodopsin I-II reaction have been measured over a wide range of temperatures (1-37C ) and pH values (4.5-8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature. The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals. All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.

Light-induced axial and radial shrinkage effects and changes of the refractive index in isolated bovine rod outer segments and disc vesicles: physical analysis of near-infrared scattering changes.

Flash-induced transients in the near-infrared scattering of bovine rod outer segments and isolated discs are investigated. Their common characteristic is the saturation at a rhodopsin bleaching of ca. 10%, which was previously described for the so-called “signalP”. The theory is based on the Rayleigh-Gans-approximation and on a cylindrical particle shape. This treatment is shown to be applicable in the measured angular range (in generalθ≤30‡), in spite of the polydisperse shape of the real particles. Using the angular dependence of the relative intensity change (difference scattering curve), changes of the polarizability (refractive index) and of the particle shape can be distinguished. Model difference scattering curves are calculated for the dimensions of the rod outer segments. Static scattering measurements are used for an estimation of the average particle shape: the isolated disc samples appear to contain flat discs as well as an admixture of rod-like structures (ca. 1% of the total scattering mass); in rod outer segment preparations, a contribution of non-rodlike scattering is found which is strongly dependent on the treatment of the sample. The flash induced transients were measured using randomly oriented particles (discs and rod outer segments) and axially oriented rod outer segments. The angular dependence of the amplitude yields its difference scattering curve. On suspensions of isolated discs, which were re-loaded with the proteins extracted at low ionic strength, one single signal is observed (termedPD, first order,Τ=0.6–1.2 s). Using randomly oriented rod outer segments, a signal with complex millisecond kinetics (termed signalP) and a slow signal (termedPS, first order,Τ=5–25 s) can be distinguished kinetically. In the axially oriented rod outer segments, theP-signal splits into a fast axial (10 ms) and a slower radial component (50–100 ms). The slow signalPS observed in ROS and the signalPD in discs have one common physical interpretation as local changes of the polarizability, directly observed in light-scattering as a change of the refractive index. The fast signalP in ROS, however, has no detectable local component but represents a pure shrinkage effect. On the axially oriented system, this shrinkage turns out to be axial and radial with different kinetics. Only rough estimations for the relative shrinkage effects and refractive index changes can be given. One obtains for 1% rhodopsin bleaching:δn/n≈10−4,δL/L≈10−2,δR/R≈5×10−4. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated from the radial shrinkage as ca. 300 å2. This high value is discussed in relation to the binding of rhodopsin to the GTP-binding protein which is involved in comparable effects described by Kühn et al. (1981). According to our data, a chemical binding process in milliseconds is only indicated in the isolated disc; in the closed disc stack of the rod outer segment, only weak (fast) local interactions are consistent with the difference scattering data. A turn or lift of the GTPase would better satisfy this condition and explain the above high value for the individual shrinkage effect.

Temperature dependence of G-protein activation in photoreceptor membranes. Transient extra metarhodopsin II on bovine disk membranes

The thermal activation barrier of guanosine triphosphate dependent dissociation of the light-induced rhodopsin-G-protein complex has been determined using a spectroscopic technique (enhanced formation of metarhodopsin II). The dissociation rate has been measured in the range - 2 degrees C less than or equal to t less than or equal to 12 degrees C. The Arrhenius plot yields apparent activation energies: 166 +/- 10 kJmol-1 with 5-guanylylimidodiphosphate (GMPPNP) and 175 +/- 15 kJmol-1 with GTP. The rhodopsin-G-protein dissociation rate is linearly related to the concentration of GMPPNP in the measurable range (less than or equal to 200 microM). The data show that, at low temperature (1 degree C), the rate limiting step of G-protein activation is the bimolecular reaction between the protein and the nucleotide. This also seems to hold true for more physiological conditions as suggested by extrapolation and comparison with nucleotide exchange rates in the literature. The high activation barrier of the nucleotide exchange reaction is explained in terms of rapid endothermic preequilibrium between an inactive and an exchanging state of the rhodopsin-G-protein complex.