Cyclic GMP is central to visual excitation in vertebrate retinal rod cells. Sodium channels in the plasma membrane of the outer segment are kept open in the dark by a high level of cGMP. Light closes these channels by activating an enzymatic cascade that leads to the rapid hydrolysis of cGMP. Photoexcited rhodopsin triggers transducin by catalyzing the exchange of GTP for bound GDP. The activated GTP-form of transducin then switches on the phosphodiesterase by overcoming an inhibitory constraint. The overall gain of this cascade is about 10(5). The cascade is turned off by the GTPase activity of transducin and by the action of rhodopsin kinase and arrestin. One of the challenges now is to delineate the interplay of cGMP, calcium ion, and phosphoinositides in excitation and adaptation. Transducin belongs to a family of signal-coupling proteins that includes the G proteins of the hormone-regulated adenylate cyclase cascade. The initial events in visual excitation in molluscs and arthropods are probably similar to those of vertebrates. The triggering of transducin by photoexcited rhodopsin is a recurring motif in visual transduction. The coming together of electrophysiology, biochemistry, and molecular genetics affords new opportunities in unraveling the molecular mechanism of visual transduction.

Cyclic GMP cascade of vision.

Receptor-effector coupling by G proteins

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP-GDP exchange on the G protein alpha-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.

Heterotrimeric G protein activation by G-protein-coupled receptors

Structure of bovine rhodopsin in a trigonal crystal form

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1016/j.febslet.2009.10.024

Membrane protein assembly into Nanodiscs

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2882%2980266-4

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

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2881%2980618-7

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

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 a2. 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 Kuhn 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.

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.

Using axially oriented rod outer segments, the P-signal splits into a fast axial (10 ms) and a slower radial signal (100 ms). A comparison of their angular dependence with calculated model curves yields,A{or a bleaching of I% rhodopsin, an axial shrinkage of 0.5% ~ -s ~ 1.5%. The radial signal is also interpreted by a (radial) contraction and estimated as 0.1% ~ $ 0.3%. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated as 200-600 ~2.

D. Emeis

Papers

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

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

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.

Physical interpretation of fast flash-induced changes in the near-infrared scattering of isolated bovine rod cell organelles