Vasily Kerov

Transducin Activation State Controls Its Light-dependent Translocation in Rod Photoreceptors

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-α subunit (Gtα)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtαQ200L. An illumination threshold for the Gtα movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtαQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtα, the GtαQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtαQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtα, and apparently, do not require Gtα interaction with RGS9 and PDE6.

Dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2

Mutations in the gene encoding Cav1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav1.4 channels, there are defects in the development of “ribbon” synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav1.4 channels. Whether defects in PR synapse development due to altered Cav1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav1.4-selective antibodies, we found that Cav1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav1.4 activation is either enhanced (Cav1.4I756T) or inhibited (CaBP4 KO), the initial stages of PR synaptic ribbon formation are largely unaffected. However, after postnatal day 13, many PR ribbons retain the immature morphology. This synaptic abnormality corresponds in severity to the defect in synaptic transmission in the adult mutant mice, suggesting that lack of sufficient mature synapses contributes to vision impairment in Cav1.4I756T and CaBP4 KO mice. Our results demonstrate the importance of proper Cav1.4 function for efficient PR synapse maturation, and that dysregulation of Cav1.4 channels in CSNB2 may have synaptopathic consequences.

Unique transducins expressed in long and short photoreceptors of lamprey Petromyzon marinus

Lampreys represent the most primitive vertebrate class of jawless fish and serve as an evolutionary model of the vertebrate visual system. Transducin-alpha (G alpha(t)) subunits were investigated in lamprey Petromyzon marinus in order to understand the molecular origins of rod and cone photoreceptor G proteins. Two G alpha(t) subunits, G alpha(tL) and G alpha(tS), were identified in the P. marinus retina. G alpha(tL) is equally distant from cone and rod G proteins and is expressed in the lampreys long photoreceptors. The short photoreceptor G alpha(tS) is a rod-like transducin-alpha that retains several unique features of cone transducins. Thus, the duplication of the ancestral transducin gene giving rise to rod transducins has already occurred in the last common ancestor of the jawed and jawless vertebrates.

Characterization of human cone phosphodiesterase-6 ectopically expressed in Xenopus laevis rods.

PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone- and rod-specific PDE6 γ-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.

Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2

Mutations in the CACNA1F gene encoding the Cav1.4 Ca2+ channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family.1 Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation—as seen in the IT gain-of-function mutant2—may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB.

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

N-terminal acylation of the α-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein α-subunit, transducin (Gαt), we generated transgenic mice that expressed a mutant Gαt lacking N-terminal acylation sequence (GαtG2A). Rods expressing GαtG2A showed a severe defect in transducin cellular localization. In contrast to native Gαt, which resides in the outer segments of dark-adapted rods, GαtG2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing GαtG2A at 5–6% of the Gαt levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by GαtG2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of GαtG2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of Gαt membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

Phototransduction in a Transgenic Mouse Model of Nougaret Night Blindness

The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-α subunit (Tα). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing TαG38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in TαG38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca2+ current, suggesting interactions of transducin with the synaptic machinery.

Diffusion and light-dependent compartmentalization of transducin.

Diffusion and light-dependent compartmentalization of transducin are essential for phototransduction and light adaptation of rod photoreceptors. Here, transgenic Xenopus laevis models were designed to probe the roles of transducin/rhodopsin interactions and lipid modifications in transducin compartmentalization, membrane mobility, and light-induced translocation. Localization and diffusion of EGFP-fused rod transducin-α subunit (Gα(t1)), mutant Gα(t1) that is predicted to be N-acylated and S-palmitoylated (Gα(t1)A3C), and mutant Gα(t1) uncoupled from light-activated rhodopsin (Gα(t1)-Ctα(s)), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to Gα(t1), Gα(t1)A3C and Gα(t1)-Ctα(s) were correctly targeted to the rod outer segments in the dark, however the light-dependent translocation of both mutants was markedly impaired. Our analysis revealed a moderate acceleration of the lateral diffusion for the activated Gα(t1) consistent with the diffusion of the separated Gα(t1)GTP and Gβ(1)γ(1) on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gα(t1)GTP with a single lipid anchor and heterotrimeric Gα(t1)β(1)γ(1) or Gα(t1)-Ctα(s)β(1)γ(1) with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the Gα(t1)A3C mutant apparently caused by its stable two lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of Gα(t1)β(1)γ(1).

Photoreceptor inner and outer segments.

Photoreceptors are exquisitely adapted to transform light stimuli into electrical signals that modulate neurotransmitter release. These cells are organized into several compartments including the unique outer segment (OS). Its whole function is to absorb light and transduce this signal into a change of membrane potential. Another compartment is the inner segment where much of metabolism and regulation of membrane potential takes place and that connects the OS and synapse. The synapse is the compartment where changes in membrane potentials are relayed to other neurons in the retina via release of neurotransmitter. The composition of the plasma membrane surrounding these compartments varies to accommodate their specific functions. In this chapter, we discuss the organization of the plasma membrane emphasizing the protein composition of each region as it relates to visual signaling. We also point out examples where mutations in these proteins cause visual impairment.