G-Protein βγ-Complex Is Crucial for Efficient Signal Amplification in Vision

Generation of Gngt1 knock-out mice.

All experiments were performed in accordance with the policy on the Use of Animals in Neuroscience Research and were approved by the Saint Louis University Institutional Animal Care and Use Committee and the Washington University Animal Studies Committee. Unless otherwise specified, all mice were age-matched 2- to 3-month-old littermates of either sex; they were kept under the standard 12 h dark/light cycle and dark-adapted overnight before all experiments.

The mouse rod Gtγ gene, Gngt1, was isolated and mapped by screening the mouse phage library. It contains three exons and two introns (Hurley et al., 1984; Yatsunami et al., 1985; Tao et al., 1993; Scherer et al., 1996; Downes and Gautam, 1999) (see Fig. 1A). The targeting construct was designed to replace all three exons with a Neo cassette to eliminate the coding region of Gtγ. The conventional targeting vector was constructed by using a 1.6 kb DNA fragment as the short homology arm (SA). It was amplified by PCR using primers located 1.9 and 0.2 kb upstream of exon 1. SA was subcloned at the 5′-end of the Neo cassette in the 5′–3′ orientation using MluI sites. The long homology arm (LA), a 7.3 kb XbaI fragment isolated from a lambda phage clone, was inserted at the 3′-end of the Neo cassette in the 5′–3′ orientation using AvrII sites. The targeting vector was confirmed by restriction analysis and sequencing. This transgenic design is notably different from the commercially available Deltagen Gngt1^−/− mouse (Deltagen; target ID 408), in which Gngt1 was targeted by a gene trap replacement of a part of the Gtγ coding sequence (amino acids 17–44) and intron 2 by the IRES-LacZ-Neo cassette.

The Gngt1 knock-out construct was electroporated into the 129 strain of ES cells, and G418-resistant clones were identified by PCR, DNA sequencing, and Southern blotting (inGenious Targeting Laboratory). Positive clones were injected into blastocysts to generate chimeric mice. Germline transmission in F₁ and in subsequent generations derived by crossings with C57BL/6 was confirmed by PCR using primers A1/N1 for the 1.8 kb knock-out (KO) allele and WTi1/WTi2 for the 460 bp wild-type (WT) allele (data not shown). The forward A1 primer (5′-GGAGAACACTCATGGAGAAGCTC-3′) was just outside of SA, and the reverse N1 primer (5′-CCAGAGGCCACTTGTGTAGC-3′) was within the Neo gene. The forward WTi1 primer (5′-GTAAGTGCAAAGCAGAGGCATGGGCTGCCTGTGGGCTC-3′) was inside intron 1, and the reverse WTi2 primer (5′-CCCGATCCAAGTGTGGCTCTTTGCCTGTTTTGGTACGAC-3′) was inside intron 2.

Antibodies and Western blotting.

Rabbit antibodies sc-389-Gtα₁, sc-390-Gtα₂, sc-380-Gβ₂, sc-381-Gβ₃, sc-374-Gγ₂, sc-375-Gγ₃, sc-376-Gγ₅, sc-377-Gγ₇, sc-15382-rhodopsin, sc-28850-phosducin, as well as goat antibodies sc-26776-Gγ₄, sc-8143-RGS9, and mouse antibodies sc-8004-GRK1, sc-73044-SNAP25 were from Santa Cruz Biotechnology. Rabbit antibodies against Gγ_c and PDEα, PDEβ, and PDEγ were from CytoSignal Research Products. Rabbit antibodies against GCAP1, GCAP2, and retGC1 were a gift from A. M. Dizhoor (Pennsylvania College of Optometry, Elkins Park, PA). Rabbit antibodies against M-opsin and S-opsin were a gift from C. M. Craft (Zhu et al., 2003) (Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, University of Southern California, Los Angeles, CA). Rabbit antibodies against Gβ₁ and Gγ₁ were a gift from N. Gautam (Washington University, St. Louis, MO). Rabbit Gβ₅ antibody was a gift from W. F. Simonds (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Mouse antibody for rod arrestin was a gift from W. C. Smith (University of Florida, Gainesville, FL). Rabbit antibody against PhLP was a gift from B. M. Willardson (Brigham Young University, Provo, UT). Rabbit antibody AB5585-recoverin was from Millipore. Secondary HRP antibodies were from Jackson ImmunoResearch Laboratories. Blots were developed using Pierce Femto Supersignal kit. Signal intensity of the protein bands on x-ray film was quantified by densitometry using Image Gauge (FujiFilm).

Light microscopy, electron microscopy, and immunohistochemistry.

For immunolabeling, eyes were fixed in freshly prepared 0.1 m phosphate buffer, pH 7.4, containing 2% paraformaldehyde and 0.1% glutaraldehyde and embedded in LR White. Semithin 0.5 μm sections were cut in the dorsal-to-ventral direction through the optic nerve and immunostained essentially as previously described (Naash et al., 2004) followed by silver intensification using an IntenSE M Silver Enhancement Kit (GE Healthcare). For electron microscopy, ultrathin 0.1 μm sections were picked up on uncoated 75/300 mesh nickel grids, stained with uranyl acetate and lead citrate, and exposed to OsO₄ vapor for 30 min.

For measurements of rod outer segment (ROS) length, the embedded blocks were sectioned in the dorsal-to-ventral direction through the optic nerve. Twenty independent measurements were made starting at ∼500 μm from the edge of the optic nerve head on both sides with 2 μm steps between individual measurements, and the mean and SEM values were calculated for each specimen.

Protein quantification and transducin membrane partitioning.

Retinas and ROS disk preparations used for Western blotting were from 2-month-old mice. Dark-adapted mouse ROS disc membranes were prepared from 50 to 150 mouse retinas, as previously described (Papermaster and Dreyer, 1974). Purified ROS disk membrane pellets contained only membrane-bound transducin subunits. They were aliquoted and stored at −80°C until protein quantification or biochemical experiments. Soluble transducin fraction was lost during the ROS disk membrane purification procedure and thus was not considered in additional analysis. Contamination by the inner segment marker, cytochrome c, was undetectable. Bovine Gtα and Gtβγ subunits were purified and total ROS disk membrane protein and rhodopsin concentration were measured as previously described (Kisselev, 2007). Using rhodopsin or total ROS disk membrane protein measurements as loading controls produced similar results.

For partitioning experiment, R*-Gt binding measurements in fully bleached ROS disk membranes were performed as described previously (Kisselev, 2007), with the following modifications: mouse ROS disk membrane pellets were resuspended at 3 μm rhodopsin in 10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm MgCl₂, 1 mm DTT, and 0.1 mm PMSF, to establish a new equilibrium between the membrane and soluble Gt. After light activation, samples were incubated on ice for 10 min, and supernatant and pellet were separated by centrifugation at 100,000 × g at 4°C for 10 min in a TLA-100.3 rotor. Gtα content in both fractions was analyzed by quantitative immunoblotting.

Electroretinography.

Animals were dark-adapted overnight and anesthetized by subcutaneous injection of ketamine (80 mg/kg) and xylazine (15 mg/kg). Pupils were dilated with 1% atropine sulfate. During testing, a heating pad controlled by a rectal temperature probe maintained body temperature at 37–38°C. Full-field ERGs were recorded using a UTAS-E3000 apparatus (LKC Technologies) and platinum corneal electrodes, as described (Brantley et al., 2008; Kolesnikov et al., 2010). Reference and ground electrode needles were inserted under the skin at the skull and the tail, respectively. Test flashes of 15–650 μs white light were applied either in darkness (scotopic conditions) or in the presence of steady background illumination (200 cd m⁻²), after 5 min adaptation to the background light (photopic conditions). Responses from several trials were averaged and the intervals between trials were adjusted so that responses did not decrease in amplitude over the series of trials for each step. The recorded responses were bandpass filtered at 0.05–1500 Hz.

Single-cell electrophysiology.

In contrast to the previously characterized Deltagen Gngt1^−/− model (Lobanova et al., 2008), suction recordings could be performed easily from the rods of our 2- to 3-month-old Gngt1^−/− mice because of the lack of early retinal degeneration. Animals were dark-adapted overnight and the retinas were removed, chopped into small pieces, and transferred to a perfusion chamber. A single rod outer segment was drawn into a glass microelectrode filled with solution containing 140 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl₂, 1.2 mm CaCl₂, 3 mm HEPES, pH 7.4, 0.02 mm EDTA, and 10 mm glucose. The perfusion solution contained 112.5 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl₂, 1.2 mm CaCl₂, 10 mm HEPES, pH 7.4, 20 mm NaHCO₃, 3 mm Na succinate, 0.5 mm Na glutamate, 0.02 mm EDTA, and 10 mm glucose. The perfusion solution was bubbled with 95% O₂/5% CO₂ mixture and heated to 37–38°C.

Light stimulation was applied by 20 ms test flashes of calibrated 500 nm light. Photoresponses were amplified, low-pass filtered (30 Hz, 8-pole Bessel), and digitized (1 kHz). Dominant recovery time constant (τ_D) was determined from supersaturating flashes (Pepperberg et al., 1992), using a 10% criterion for photocurrent recovery from saturation. The amplification of the rod phototransduction cascade was evaluated from test flash intensities that produced identical rising phases of dim flash responses. This approach was preferred to the Lamb and Pugh determination of the amplification constant (Pugh and Lamb, 1993) because of the relatively long duration of test flashes and the effect of low-pass filtering on the response front.

Spatial contrast sensitivity measured from optomotor responses.

Spatial contrast visual sensitivity of age-matched 2- to 3-month-old mice was measured using a two-alternative forced-choice protocol (Umino et al., 2008). The Optomotry system (Cerebral Mechanics) consisted of a square array of four computer monitors with a pedestal in the center where the mouse was placed. An infrared-sensitive television camera and a round array of six infrared LEDs mounted above the animal were used to observe the mouse but not the monitors. Using a staircase paradigm, rotating stimuli (sine wave vertical gratings) were applied on the monitors where they formed a virtual cylinder around the mouse (Prusky et al., 2004). Mice responded to the stimuli by reflexively rotating their head in the corresponding direction. Contrast sensitivity was defined as the inverse of contrast threshold for optomotor responses. Responses were measured over a range of background light intensities, from −6.25 to 1.85 log cd m⁻². Background monitor luminance was controlled by neutral density film filters (E-Color 211 0.9 ND; Rosco Laboratories). Temporal frequency (f_t) was fixed at its optimal value of 0.75 Hz for all background conditions. Spatial frequency (f_s) was varied in the range of 0.014–0.481 cyc/deg, and speed of the stimuli was adjusted based on the following equation: f_t = s_p · f_s (Umino et al., 2008). For determination of maximal contrast sensitivity under each condition, data were fitted with mouse contrast sensitivity model (Umino et al., 2008), using parameters adjusted for best fit (r² > 0.8). All data were analyzed using independent two-tailed Student's t test, with accepted significance level of p < 0.05.

Mathematical modeling of phototransduction.

A mathematical model of phototransduction (Kuzmin et al., 2004) was used. This model includes all firmly established biochemical mechanisms of phototransduction and its regulation by calcium feedback. Basic equations of the model are similar to those used in many previous works (Hamer, 2000a,b; Nikonov et al., 2000; Hamer et al., 2003, 2005). However, our treatment of Ca²⁺ regulation and Ca²⁺ turnover differs slightly from that used before. Therefore, we present here the full set of equations comprising the model.

Number of active rhodopsin molecules, R*, is determined by a balance between its generation by light, I(t), and quenching by phosphorylation with rhodopsin kinase. We omit the detailed description of Ca²⁺ regulation of rhodopsin kinase via recoverin (Hamer et al., 2003, 2005) and instead use an empirical Hill-like relationship (the term in parentheses on right side of Eq. 1) (Calvert et al., 1998) as follows:

Here, k_Rmin and k_Rmax are minimum and maximum rate constants of phosphorylation (in seconds⁻¹) at very high and zero Ca²⁺ concentrations, respectively. K_CaR is the half-saturating Ca²⁺ concentration, and n_CaR is the Hill's coefficient of regulation.

Number of activated phosphodiesterase molecules E* is given by the following: where ν_RE is the rate of PDE activation by single R* (in seconds⁻¹), and k_E is the rate constant of E* turnoff (in seconds⁻¹).

cGMP turnover is described by the following: where α(t) is the rate of cGMP production by guanylate cyclase, and β(t) is the rate of its hydrolysis by phosphodiesterase. Here, cGMP concentration is expressed in moles · liter⁻¹, and α(t) and β(t), in moles · liter⁻¹ · second⁻¹.

Guanylate cyclase activity is under calcium control, in the form similar to that for R* turnoff as follows: Notice that, in this formulation, like in the study by Nikonov et al. (2000) (Eq. A10), the extent of guanylate cyclase regulation is limited by the range between α_max and α_min, in contrast to most recent models (Burns et al., 2002; Hamer et al., 2003, 2005) that assume infinite regulation range (α_min = 0).

The rate of cGMP hydrolysis is as follows:

Here, β_Dark is the steady PDE activity in darkness, and the second term in parentheses yields light-induced activity. k_cat is the catalytic activity of a single light-activated PDE subunit (in seconds⁻¹), whereas ROS cytoplasmic volume V_cyto and Avogadro's number N_Av convert the number of photoactivated PDE molecules into concentration. Hydrolysis of cGMP is supposed to proceed in accordance with Michaelis kinetics, with the half-saturating cGMP concentration K_m. Again, we do not make the simplifying assumption cG(t) ≪ K_m common in recent models.

The ROS membrane current is a sum of two components, the current flowing through cGMP-gated channels j_cG(t) and the current carried by the Ca,Na/K exchanger j_ex(t): where j_cGmax is maximum current at saturating cGMP concentrations, K_cG is a half-saturating concentration, and n_cG is the Hill's coefficient of the regulation of the channels.

Free Ca²⁺ turnover is described by the following: Here, f_Ca is the fraction of the ROS current carried by Ca²⁺, j_ex is Ca²⁺ extrusion current carried by Ca,K/Na exchanger, and ℑ is Faraday's number [cf. Hamer et al. (2005), their Eq. A9]. In accordance with experimental data on amphibian rods (McCarthy et al., 1996; Younger et al., 1996; Govardovskii and Kuzmin, 1999), ROSs are supposed to contain a two-component Ca²⁺ buffer. One of the components exchanges with free Ca²⁺ quickly, so its effect on free Ca²⁺ turnover can simply be characterized by its buffering power, FB (Lagnado et al., 1992). The second, slowly exchangeable component is characterized by its binding capacity B_max, and two rate constants, k₊₁ for binding and k₋₁ for releasing Ca²⁺. Ca_Bslow(t) is the concentration of calcium bound to slow buffer and is described by the following:

In the present paper, we set B_max = 0 (see Table 2). With properly chosen FB, this does not significantly affect the quality of fitting of flash responses but reduces the number of free model parameters.

The exchanger current is described in Michaelis' manner (Cervetto et al., 1989) as follows: The parameters of the model were chosen to be within empirical biochemical and biophysical values when such data were available (Tables 1, 2). This provided a great flexibility of fitting, allowing virtually perfect simulation of photoresponses (coefficient of correlation between experimental and model traces r > 0.999 in wild-type and Gngt1^−/− rods, and r > 0.997 in Gngt1^+/− rods). However, our goal was not to produce the best fit of the experimental data, but rather to determine what parameters of the phototransduction cascade must be modified to account for the effects of Gtγ deletion. Thus, a group of parameters that was unlikely to be affected by the lack of Gtγ was kept constant among wild-type, Gngt1^+/−, and Gngt1^−/− rods. These included properties of the ROS plasma membrane, namely, surface density, ionic selectivity, and affinity to cGMP of the cGMP-gated channels, and properties of the Ca²⁺,K⁺/Na⁺ exchanger. Since the dark current was virtually unchanged in genetically manipulated rods, the above assumptions imply that the dark concentrations of cGMP and Ca²⁺ also remained constant. Although a constant dark cGMP level does not necessary mean that dark guanylate cyclase and phosphodiesterase activities remained unchanged, the two parameters were fixed as well. Furthermore, parameters of the Ca²⁺ modulation of the cascade components (fold regulation, affinities, and Hill's coefficients in Eqs. 1 and 4) were also kept constant.

For proper comparison with experimental responses, model curves were Gauss-filtered, with smoothing window of 23 ms equivalent to experimental 30 Hz Bessel filter.