Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3'',5'' cyclic monophosphate in the salamander rod outer segment.

Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.     

Characterization of guanylate cyclase activity in single retinal rod outer segments

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.     

Diffusion coefficient of cyclic GMP in salamander rod outer segments estimated with two fluorescent probes.

Experiments have demonstrated that single photoisomerizations in amphibian and primate rods can cause the suppression of 3-5% of the dark circulating current at the response peak (Baylor, D. A., T. D. Lamb, and K. W. Yau. 1979. J. Physiol. (Lond.). 288:613-634; Baylor, D. A., B. J. Nunn, and J. L. Schnapf. 1984. J. Physiol. (Lond.). 357:575-607). These results indicate that the change in [cGMP] effected by a single isomerization must spread longitudinally over at least the corresponding fractional length of the outer segment. The effective longitudinal diffusion coefficient, Dx, of cGMP is thus an important determinant of rod sensitivity. We report here measurements of the effective longitudinal diffusion coefficients, Dx, of two fluorescently labeled molecules: 5/6-carboxyfluorescein and 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate, introduced into detached outer segments via whole-cell patch electrodes. For these compounds, the average time for equilibration of the entire outer segment with the patch pipette was approximately 6 min. Fluorescence images of rods were analyzed with a one-dimensional diffusion model that included limitations on transfer between the electrode and outer segment and the effects of intracellular binding of the dyes. The analyses yielded estimates of Dx of 1.9 and 1.0 microns 2.s-1 for the two dyes. It is shown that these results place an upper limit on Dx for cGMP of 11 microns2.s-1. The actual value of Dx for cGMP in the rod will depend on the degree of intracellular binding of cGMP. Estimates of the effective buffering power for cGMP in the rod at rest range from two to six (Lamb and Pugh, 1992; Cote and Brunnock, 1993). When combined with these estimates, our results predict that for cGMP itself, Dx falls within the range of 1.4-5.5 microns 2.s-1.     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Calcium diffusion coefficient in rod photoreceptor outer segments.

Calcium (Ca(2+)) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca(2+) enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na(+)/Ca(2+),K(+) exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca(2+). This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca(2+) concentration propagates through the rod outer segment depends on the Ca(2+) diffusion coefficient. We have used the fluorescent Ca(2+) indicator fluo-3 and confocal microscopy to measure the profile of the Ca(2+) concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 +/- 1 microm(2)s(-1) for the radial Ca(2+) diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L.Lagnado, L. Cervetto, and P.A. McNaughton, 1992, J. Physiol. 455:111-142) and with a buffering capacity of approximately 20 for rods in darkness(S. Nikonov, N. Engheta, and E.N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7-37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca(2+). Also, because of baffling by the disks, the longitudinal Ca(2+) diffusion coefficient will be in the order of 2 microm(2)s(-1), which is much smaller than the longitudinal cGMP diffusion coefficient (30-60 microm(2)s(-1); ). Therefore, the longitudinal decline of Ca(2+) lags behind the longitudinal spread of excitation by cGMP.     

Calcium feedback and sensitivity regulation in primate rods.

Membrane current was recorded from a single primate rod with a suction pipette while the cell was bath perfused with solutions maintained at a temperature of approximately 38 degrees C. A transient inward current was observed at the onset of bright illumination after briefly exposing the outer segment in darkness to Ringer's (Locke) solution containing 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cGMP phosphodiesterase. After briefly removing external Na+ from around the outer segment in darkness, a similar current was observed upon Na+ restoration in bright light. By analogy to amphibian rods, this inward current was interpreted to represent the activity of an electrogenic Na(+)-dependent Ca2+ efflux, which under physiological conditions in the light is expected to reduce the free Ca2+ in the outer segment and provide negative feedback (the "Ca2+ feedback") to the phototransduction process. The exchange current had a saturated amplitude of up to approximately 5 pA and a decline time course that appeared to have more than one exponential component. In the absence of the Ca2+ feedback, made possible by removing the Ca2+ influx and efflux at the outer segment using a 0 Na(+)-0 Ca2+ external solution, the response of a rod to a dim flash was two to three times larger and had a longer time to peak than in physiological solution. These changes can be approximately accounted for by a simple model describing the Ca2+ feedback in primate rods. The dark hydrolytic rate for cGMP was estimated to be 1.2 s-1. The incremental hydrolytic rate, beta*(t), activated by one photoisomerization was approximately 0.09 s-1 at its peak, with a time-integrated activity, integral of beta*(t)dt, of approximately 0.033, both numbers being derived assuming spatial homogeneity in the outer segment. Finally, we have found that primate rods adapt to light in much the same way as amphibian and other mammalian rods, such as showing a Weber-Fechner relation between flash sensitivity and background light. The Ca2+ feedback model we have constructed can also explain this feature reasonably well.     

The cGMP-phosphodiesterase and its contribution to sensitivity regulation in retinal rods

We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.     

Effects of cyclic GMP on the kinetics of the photocurrent in rods and in detached rod outer segments

We investigated the effects of high concentrations of cytoplasmic cyclic GMP on the photocurrent kinetics and light sensitivity of the tiger salamander rod both in intact cells and in detached outer segments. Photoreceptors were internally perfused with cGMP by applying patch pipettes containing cGMP to the inner or outer segment. Large increases in the concentration of cGMP in the outer segment cytoplasm were achieved only when the patch pipette was applied directly to the outer segment. The dark-current amplitude increased with increasing cGMP concentrations up to approximately 1,400 pA. Internal perfusion with 5.0 mM cGMP introduced a delay of 1-3 s in the photocurrent. The magnitude of the delay was inversely proportional to the light intensity. In addition, the photocurrent time course was slowed down and the light sensitivity, measured 1 s after the flash, was decreased approximately 100-fold when compared with that of the intact cell. The observed effects of cGMP were compared with those predicted by a model that assumes that the initial photocurrent time course is determined by the kinetics of the light-activated phosphodiesterase (PDE) and the cGMP dependence of the light-sensitive channels. At high concentrations of cGMP, the experimental data were similar to those predicted by the model and based on the known biochemical properties of the light-activated PDE and cGMP-activated channels.     

A 26 kd calcium binding protein from bovine rod outer segments as modulator of photoreceptor guanylate cyclase.

The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key events leading to the reopening of cGMP-gated channels after photoexcitation. Guanylate cyclase activity in vertebrate rod outer segments is dependent on the free calcium concentration. The basal activity of the enzyme observed at high concentrations of free calcium (greater than 0.5 microM) increases when the free calcium concentration is lowered into the nanomolar range (less than 0.1 microM). This effect of calcium is known to be mediated by a soluble calcium-sensitive protein in a highly cooperative way. We here show that this soluble protein, i.e. the modulator of photoreceptor guanylate cyclase, is a 26 kd protein. Reconstitution of the purified 26 kd protein with washed rod outer segment membranes containing guanylate cyclase revealed a 3- to 4-fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 microM to 4 nM. Guanylate cyclase in whole rod outer segments was stimulated 10-fold in the same calcium range. The activation process in the reconstituted system was similar to the one in the native rod outer segment preparation, it showed a high cooperativity with a Hill coefficient n between 1.4 and 3.5. The half-maximal activation occurred between 110 and 220 nM free calcium. The molar ratio of the modulator to rhodopsin is 1:76 +/- 32. The protein is a calcium binding protein as detected with 45Ca autoradiography. Partial amino acid sequence analysis revealed a 60% homology to visinin from chicken cones.     

Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean +/- SD): membranes contain about one cGMP binding site per 130 rhodopsin molecules; the concentration of free ligand for half saturation is 2.0 +/- 0.6 microM; the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 +/- 0.5; half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.     

Longitudinal diffusion in retinal rod and cone outer segment cytoplasm: the consequence of cell structure

Excitation signals spread along photoreceptor outer segments away from the site of photon capture because of longitudinal diffusion of cGMP, a cytoplasmic second messenger. The quantitative features of longitudinal diffusion reflect the anatomical structure of the outer segment, known to be profoundly different in rod and cone photoreceptors. To explore how structural differences affect cytoplasmic diffusion and to assess whether longitudinal diffusion may contribute to the difference in signal transduction between photoreceptor types, we investigated, both theoretically and experimentally, the longitudinal diffusion of small, hydrophilic molecules in outer segments. We developed a new theoretical analysis to explicitly compute the longitudinal diffusion constant, Dl, in terms of outer segment structure. Using time-resolved fluorescence imaging we measured Dl of Alexa488 and lucifer yellow in intact, single cones and validated the theoretical analysis. We used numerical simulations of the theoretical model to investigate cGMP diffusion in outer segments of various species. At a given time interval, cGMP spreads further in rod than in cone outer segments of the same dimensions. Across all species, the spatial spread of cGMP at the peak of the dim light photocurrent is 3-5 microm in rod outer segments, regardless of their absolute size. Similarly the cGMP spatial spread is 0.7-1 microm in cone outer segments, independently of their dimensions.     

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.     

Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods

The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of ∼200 seconds, but tip sensitivity recovered more slowly with a time constant of ∼680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is ∼1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.     

Helminth and leech community structure in tadpoles and caudatan larvae of two amphibian species from Western Nebraska

Currently no comparative studies exist on helminth and leech community structure among sympatric anuran tadpoles and salamander larvae. During June-August 2007-2009, we examined 50 bullfrog tadpoles, Rana catesbeiana , 50 barred tiger salamander larvae, Ambystoma mavortium , and 3 species of snails from Nevens Pond, Keith County, Nebraska for helminth and leech infections. The helminth and leech compound community of this larval amphibian assemblage consisted of at least 7 species, 4 in bullfrog tadpoles and 4 in barred tiger salamander larvae. Bullfrog tadpoles were infected with 2 species of nematodes ( Gyrinicola batrachiensis and Spiroxys sp.) and 2 types of metacercariae ( Telorchis sp. and echinostomatids), whereas barred tiger salamander larva were infected with 1 species of leech ( Placobdella picta ), 2 species of adult trematodes ( Telorchis corti and Halipegus sp.), and 1 species of an unidentified metacercaria. The component community of bullfrog tadpoles was dominated by helminths acquired through active penetration, or incidentally ingested through respiratory currents, or both, whereas the component community of larval salamanders was dominated by helminths acquired through ingestion of intermediate hosts (χ2 = 3,455.00, P < 0.00001). Differences in amphibian larval developmental time (2-3 yr for bullfrog tadpoles versus 2-5 mo for salamander larvae), the ephemeral nature of intermediate hosts in Nevens Pond, and the ability of bullfrog tadpole to eliminate echinostome infections had significant effects on mean helminth species richness among amphibian species and years (t = 12.31, P < 0.0001; t = 2.09, P = 0.04). Differences in herbivorous and carnivorous diet and time to metamorphosis among bullfrog tadpoles and barred tiger salamander larvae were important factors in structuring helminth communities among the larval stages of these 2 sympatric amphibian species, whereas size was important in structuring helminth and leech communities in larval salamanders, but not in bullfrog tadpoles.     

Modulation by internal protons of native cyclic nucleotide-gated channels from retinal rods

Ion channels directly activated by cyclic nucleotides are present in the plasma membrane of retinal rod outer segments. These channels can be modulated by several factors including internal pH (pH(i)). Native cyclic nucleotide-gated channels were studied in excised membrane patches from the outer segment of retinal rods of the salamander. Channels were activated by cGMP or cAMP and currents as a function of voltage and cyclic nucleotide concentrations were measured as pH(i) was varied between 7.6 and 5.0. Increasing internal proton concentrations reduced the current activated by cGMP without modifying the concentration (K(1/2)) of cGMP necessary for half-activation of the maximal current. This effect could be well described as a reduction of single-channel current by protonation of a single acidic residue with a pK(1) of 5.1. When channels were activated by cAMP a more complex phenomenon was observed. K(1/2) for cAMP decreased by increasing internal proton concentration whereas maximal currents activated by cAMP increased by lowering pH(i) from 7.6 to 5.7-5.5 and then decreased from pH(i) 5.5 to 5.0. This behavior was attributed both to a reduction in single-channel current as measured with cGMP and to an increase in channel open probability induced by the binding of three protons to sites with a pK(2) of 6.     

Mathematical model of the spatio-temporal dynamics of second messengers in visual transduction

A model describing the role of transversal and longitudinal diffusion of cGMP and Ca(2+) in signaling in the rod outer segment of vertebrates is developed. Utilizing a novel notion of surface-volume reaction and the mathematical theories of homogenization and concentrated capacity, the diffusion of cGMP and Ca(2+) in the inter-disc spaces is shown to be reducible to a one-parameter family of diffusion processes taking place on a single rod cross section; whereas the diffusion in the outer shell is shown to be reducible to a diffusion on a cylindrical surface. Moreover, the exterior flux of the former serves as a source term for the latter, alleviating the assumption of a well-stirred cytosol. A previous model of visual transduction that assumes a well-stirred rod outer segment cytosol (and thus contains no spatial information) can be recovered from this model by imposing a "bulk" assumption. The model shows that upon activation of a single rhodopsin, cGMP changes are local, and exhibit both a longitudinal and a transversal component. Consequently, membrane current is also highly localized. The spatial spread of the single photon response along the longitudinal axis of the outer segment is predicted to be 3-5 microm, consistent with experimental data. This approach represents a tool to analyze point-wise signaling dynamics without requiring averaging over the entire cell by global Michaelis-Menten kinetics.     

Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism.

The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

Retinal photoreceptor fine structure in the red-backed salamander (Plethodon cinereus).

The retinal photoreceptors of the red-backed salamander (Plethodon cinerus) have been studied by light and electron microscopy. Rods and single cones are present in this duplex retina in a ratio of about 25:1. The photoreceptors in this amphibian species are much larger than is reported for most vertebrates. In the light-adapted state, rods reach deep into the retinal epithelial (RPE) layer. The rod outer segment is composed of discs of uniform diameter displaying several very deep incisors. The rod inner segment displays a distal elliposid of mitochondria and a short stout myoid region. Rod nuclei are electron dense and often protrude through the external limiting membrane. Rod synaptic spherules are large and display several invaginated synaptic sites as well as superficial synapses. It is felt that the rods do not undergo retinomotor movements. The cone photoreceptors are much smaller than the rods and display a tapering outer segment, an unusual modified ellipsoid and a large parabolid of glycogen in the inner segment. Cone nuclei are less electron dense than rods and are located at all levels within the outer nuclear layer. The synaptic pedicle of the cones is larger, more electron lucent and display more synaptic sites (both invaginated and superficial) than that of rods. It is felt that cone photomechanical responses are minimal.     

Direct action of cGMP on the conductance of retinal rod plasma membrane

In order to identify the intracellular transmitter in the phototransduction process in the retinal rod, the action of cGMP, 2',3'cGMP, cAMP, GMP and Ca2+ on the isolated inside-out patches of the plasma membrane of retinal rods of the frog (Rana temporaria) was studied. cGMP applied at the intracellular membrane surface markedly increased the conductance of patches. The action of cGMP took place in the absence of nucleoside triphosphates and, hence, was not mediated by protein phosphorylation. The dependence of cGMP-induced component of conductance on cGMP concentration was S-shaped, with half-saturation within 10-30 microM and a Hill coefficient of about 1.7-1.8. cAMP, 2',3'cGMP, GMP (1 mM) did not exhibit any action on the membrane. Ca2+ did not affect the patch conductance in the absence of cGMP. In the presence of cGMP, lowering Ca2+ concentration from 10(-3) to 10(-8) M decreased the cGMP-dependent component of conductance by 20-30%. The approximate value of the elementary event underlying the cGMP-induced conductance estimated from the magnitude of the variance of the cGMP-induced current is within 100-250 fS. We suppose that the cGMP-activated channels found by us provide the light-sensitive conductance of the rod plasma membrane in vivo and that cGMP is the intracellular transmitter acting in the phototransduction process.