Dynamics of cyclic GMP synthesis in retinal rods
2002; 36 (1): 81-91
In retinal rods, Ca(2+) exerts negative feedback control on cGMP synthesis by guanylate cyclase (GC). This feedback loop was disrupted in mouse rods lacking guanylate cyclase activating proteins GCAP1 and GCAP2 (GCAPs(-/-)). Comparison of the behavior of wild-type and GCAPs(-/-) rods allowed us to investigate the role of the feedback loop in normal rod function. We have found that regulation of GC is apparently the only Ca(2+) feedback loop operating during the single photon response. Analysis of the rods' light responses and cellular dark noise suggests that GC normally responds to light-driven changes in [Ca(2+)] rapidly and highly cooperatively. Rapid feedback to GC speeds the rod's temporal responsiveness and improves its signal-to-noise ratio by minimizing fluctuations in cGMP.
View details for Web of Science ID 000178295100009
View details for PubMedID 12367508
Molecular mechanism of spontaneous pigment activation in retinal cones
2002; 83 (1): 184-193
Spontaneous current and voltage fluctuations (dark noise) in the photoreceptor cells of the retina limit the ability of the visual system to detect dim light. We recorded the dark current noise of individual salamander L cones. Previous work showed that the dark noise in these cells arises from thermal activation of the visual pigment. From the temperature dependence of the rate of occurrence of elementary noise events, we found an Arrhenius activation energy E(a) of 25 +/- 7 kcal/mol (mean +/- SD). This E(a) is similar to that reported for the thermal isomerization of 11-cis retinal in solution, suggesting that the cone pigment noise results from isomerization of the retinal chromophore. E(a) for the cone noise is similar to that previously reported for the "photon-like" noise of rods, but the preexponential factor is five orders of magnitude higher. To test the hypothesis that thermal isomerization can only occur in molecules whose Schiff base linkage is unprotonated, we changed the pH of the solution bathing the cone outer segment. This had little effect on the rate of occurrence of elementary noise events. The rate was also unchanged when the cone was exposed to Ringer solution made up from heavy water, whose solvent isotope effect should reduce the probability, that the Schiff base nitrogen is naked.
View details for Web of Science ID 000176445800017
View details for PubMedID 12080111
Activation, deactivation, and adaptation in vertebrate, photoreceptor cells
ANNUAL REVIEW OF NEUROSCIENCE
2001; 24: 779-805
Visual transduction captures widespread interest because its G-protein signaling motif recurs throughout nature yet is uniquely accessible for study in the photoreceptor cells. The light-activated currents generated at the photoreceptor outer segment provide an easily observed real-time measure of the output of the signaling cascade, and the ease of obtaining pure samples of outer segments in reasonable quantity facilitates biochemical experiments. A quiet revolution in the study of the mechanism has occurred during the past decade with the advent of gene-targeting techniques. These have made it possible to observe how transduction is perturbed by the deletion, overexpression, or mutation of specific components of the transduction apparatus.
View details for Web of Science ID 000170109100025
View details for PubMedID 11520918
Modification of cyclic nucleotide-gated ion channels by ultraviolet light
JOURNAL OF GENERAL PHYSIOLOGY
2000; 116 (2): 227-252
We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.
View details for Web of Science ID 000088733200010
View details for PubMedID 10919869
Receptive-field microstructure of blue-yellow ganglion cells in primate retina
1999; 2 (10): 889-893
We examined the functional microcircuitry of cone inputs to blue-ON/yellow-OFF (BY) ganglion cells in the macaque retina using multielectrode recording. BY cells were identified by their ON responses to blue light and OFF responses to red or green light. Cone-isolating stimulation indicated that ON responses originated in short (S) wavelength-sensitive cones, whereas OFF responses originated in both long (L) and middle (M) wavelength-sensitive cones. Stimulation with fine spatial patterns revealed locations of individual S cones in BY cell receptive fields. Neighboring BY cells received common but unequal inputs from one or more S cones. Inputs from individual S cones differed in strength, indicating different synaptic weights, and summed approximately linearly to control BY cell firing.
View details for Web of Science ID 000083883200014
View details for PubMedID 10491609
Control of rhodopsin activity in vision
1998; 12: 521-525
Although rhodopsin's role in activating the phototransduction cascade is well known, the processes that deactivate rhodopsin, and thus the rest of the cascade, are less well understood. At least three proteins appear to play a role: rhodopsin kinase, arrestin and recoverin. Here we review recent physiological studies of the molecular mechanisms of rhodopsin deactivation. The approach was to monitor the light responses of individual mouse rods in which rhodopsin was altered or arrestin was deleted by transgenic techniques. Removal of rhodopsin's carboxy-terminal residues which contain phosphorylation sites implicated in deactivation, prolonged the flash response 20-fold and caused it to become highly variable. In rods that did not express arrestin the flash response recovered partially, but final recovery was slowed over 100-fold. These results are consistent with the notion that phosphorylation initiates rhodopsin deactivation and that arrestin binding completes the process. The stationary night blindness of Oguchi disease, associated with null mutations in the genes for arrestin or rhodopsin kinase, presumably results from impaired rhodopsin deactivation, like that revealed by the experiments on transgenic animals.
View details for Web of Science ID 000074777200005
View details for PubMedID 9775212
The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1998; 95 (11): 6474-6479
Recoverin is a heterogeneously acylated calcium-binding protein thought to regulate visual transduction. Its effect on the photoresponse was investigated by dialyzing the recombinant protein into truncated salamander rod outer segments. At high Ca2+ (Ca), myristoylated recoverin (Ca-recoverin) prolonged the recovery phase of the bright flash response but had less effect on the dim flash response. The prolongation of recovery had an apparent Kd for Ca of 13 microM and a Hill coefficient of 2. The prolongation was shown to be mediated by inhibition of rhodopsin deactivation. After a sudden imposed drop in Ca concentration, the effect of recoverin switched off with little lag. The myristoyl (C14:0) modification of recoverin increased its activity 12-fold, and the C12:0 or C14:2 acyl group gave similar effects. These experiments support the notion that recoverin mediates Ca-dependent inhibition of rhodopsin phosphorylation and thereby controls light-triggered phosphodiesterase activity, particularly at high light levels.
View details for Web of Science ID 000073852600112
View details for PubMedID 9600991
View details for PubMedCentralID PMC27811
Mosaic arrangement of ganglion cell receptive fields in rabbit retina
JOURNAL OF NEUROPHYSIOLOGY
1997; 78 (4): 2048-2060
The arrangement of ganglion cell receptive fields on the retinal surface should constrain several properties of vision, including spatial resolution. Anatomic and physiological studies on the mammalian retina have shown that the receptive fields of several types of ganglion cells tile the retinal surface, with the degree of receptive field overlap apparently being similar for the different classes. It has been difficult to test the generality of this arrangement, however, because it is hard to sample many receptive fields in the same preparation with conventional single-unit recording. In our experiments, the response properties and receptive fields of up to 80 neighboring ganglion cells in the isolated rabbit retina were characterized simultaneously by recording with a multielectrode array. The cells were divided into 11 classes on the basis of their characteristic light responses and the temporal structures of their impulse trains. The mosaic arrangement of receptive fields for cells of a given class was examined after the spatial profile of each receptive field was fitted with a generalized Gaussian surface. For eight cell classes the mosaic arrangement was similar: the profiles of neighboring cells approached each other at the 1-sigma border. Thus field centers were 2 sigma apart. The layout of fields for the remaining three classes was not well characterized because the fields were poorly fitted by a single Gaussian or because the cells responded selectively to movement. The 2-sigma center-center spacing may be a general principle of functional organization that minimizes spatial aliasing and confers a uniform spatial sensitivity on the ganglion cell population.
View details for Web of Science ID A1997YD81900028
View details for PubMedID 9325372
Molecular origin of continuous dark noise in rod photoreceptors
1996; 71 (5): 2553-2572
Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.
View details for Web of Science ID A1996VQ52000028
View details for PubMedID 8913594
AN ALTERNATIVE PATHWAY FOR SIGNAL FLOW FROM ROD PHOTORECEPTORS TO GANGLION-CELLS IN MAMMALIAN RETINA
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1995; 92 (23): 10658-10662
Rod signals in the mammalian retina are thought to reach ganglion cells over the circuit rod-->rod depolarizing bipolar cell-->AII amacrine cell-->cone bipolar cells-->ganglion cells. A possible alternative pathway involves gap junctions linking the rods and cones, the circuit being rod-->cone-->cone bipolar cells-->ganglion cells. It is not clear whether this second pathway indeed relays rod signals to ganglion cells. We studied signal flow in the isolated rabbit retina with a multielectrode array, which allows the activity of many identified ganglion cells to be observed simultaneously while the preparation is stimulated with light and/or exposed to drugs. When transmission between rods and rod depolarizing bipolar cells was blocked by the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), rod input to all On-center and briskly responding Off-center ganglion cells was dramatically reduced as expected. Off responses persisted, however, in Off-center sluggish and On-Off direction-selective ganglion cells. Presumably these responses were generated by the alternative pathway involving rod-cone junctions. This APB-resistant pathway may carry the major rod input to Off-center sluggish and On-Off direction-selective ganglion cells.
View details for Web of Science ID A1995TD89000044
View details for PubMedID 7479860
CONDUCTANCE AND KINETICS OF SINGLE CGMP-ACTIVATED CHANNELS IN SALAMANDER ROD OUTER SEGMENTS
JOURNAL OF PHYSIOLOGY-LONDON
1995; 483 (3): 567-582
1. The conductance and kinetics of single 3',5'-cyclic guanosine monophosphate (cGMP)-activated channels of retinal rod outer segments were studied in inside-out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two-state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s-1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s-1 and was voltage insensitive. Assuming that the open-state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady-state macroscopic current-voltage relation of multichannel patches, at saturating cGMP concentration.
View details for Web of Science ID A1995QP34200003
View details for PubMedID 7539844
CALCIUM CONTROLS LIGHT-TRIGGERED FORMATION OF CATALYTICALLY ACTIVE RHODOPSIN
1994; 367 (6460): 273-277
Background light reduces the gain of phototransduction in retinal rods so that the ability to register changes in light intensity is not prevented by saturation of the cell's response. The gain is reduced by a light-induced fall in the intracellular calcium concentration which results from blockage of Ca2+ entry through the channels closed by light and continued Ca2+ extrusion by the Na:Ca,K exchanger. Calcium seems to exert several coordinated effects on the cyclic GMP cascade: a fall in [Ca2+] stimulates cGMP synthesis, increases the affinity of the cGMP-gated channel for cGMP and accelerates rhodopsin deactivation by phosphorylation. We now report that lowering intracellular [Ca2+] reduces the catalytic rhodopsin activity produced by light. The effect is operationally equivalent to a fourfold reduction in the number of rhodopsin molecules available for activation. The reduction in gain is cooperative and half-maximal at about 35 nM Ca2+, suggesting that it is mediated by a specific Ca(2+)-binding protein. Reduced rhodopsin activity in low Ca2+ should contribute to adaptation in background light.
View details for Web of Science ID A1994MR49400057
View details for PubMedID 8121492
INTERACTIONS BETWEEN DIVALENT-CATIONS AND THE GATING MACHINERY OF CYCLIC GMP-ACTIVATED CHANNELS IN SALAMANDER RETINAL RODS
JOURNAL OF GENERAL PHYSIOLOGY
1993; 101 (1): 1-25
The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.
View details for Web of Science ID A1993KG51600001
View details for PubMedID 7679715
- SYNAPTIC CIRCUITRY OF THE RETINA AND OLFACTORY-BULB CELL 1993; 72: 139-149
CATION INTERACTIONS WITHIN THE CYCLIC GMP-ACTIVATED CHANNEL OF RETINAL RODS FROM THE TIGER SALAMANDER
JOURNAL OF PHYSIOLOGY-LONDON
1992; 449: 759-783
1. The ionic dependence of current through the 3',5'-cyclic guanosine monophosphate (cyclic GMP)-activated channels of salamander rods was studied in excised inside-out membrane patches from isolated outer segments. Voltage-clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP-induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman-Hodgkin-Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl- was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl- permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current-voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP-induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP-induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1-2 mM range. Block by Mg2+ was voltage dependent, suggesting that the binding site was within the channel's transmembrane electric field. Raising the Mg2+ concentration on the external surface of the patch increased the apparent dissociation constant of block by internal Mg2+, as expected if external and internal Mg2+ compete for the same binding site. 7. Block by internal Ca2+ had an opposite and weaker voltage dependence than block by internal Mg2+. 8. In symmetrical solutions containing both Na+ and Mg2+ the outward rectification was more pronounced than in solutions containing Na+ alone. In solutions thought to be close to physiological the outward patch current increased e-fold for a depolarization of 24-30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1992HN24900042
View details for PubMedID 1381754
CYCLIC GMP-ACTIVATED CHANNELS OF SALAMANDER RETINAL RODS - SPATIAL-DISTRIBUTION AND VARIATION OF RESPONSIVENESS
JOURNAL OF PHYSIOLOGY-LONDON
1992; 448: 257-274
1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns-2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns-2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1992HJ10000013
View details for PubMedID 1375637
RAPID CHARGE MOVEMENTS AND PHOTOSENSITIVITY OF VISUAL PIGMENTS IN SALAMANDER RODS AND CONES
JOURNAL OF PHYSIOLOGY-LONDON
1991; 442: 761-780
1. Photosensitivities of visual pigments were determined by measuring early receptor currents (ERCs) in voltage-clamped photoreceptors from larval salamanders. 2. As expected from previous work of others, the ERC elicited by a brief flash consisted of a rapid inward component followed by a larger and slower outward component. The magnitude of the outward component corresponded to the movement of about 0.18 electronic charge across the membrane per photoisomerization. 3. The time course of the ERC was independent of the flash intensity, the flash wavelength and the magnitude of the response. The outward component of the cone ERC declined about twice as rapidly as the outward component of the rod ERC.. 4. The amplitude of the ERC decreased as successive flashes bleached the cell's pigment. Using the proportional relation between the size of the ERC and the number of pigment molecules photoisomerized, photosensitivities of the native A2 pigments in rods, red-sensitive cones, blue-sensitive cones and UV-sensitive cones were determined. Calculated solution photosensitivities for rhodopsin, red-sensitive and blue-sensitive cone pigments were not significantly different and the average value for all three pigments at their respective absorption maxima was (7.3 +/- 1.6) x 10(-9) micron 2 molecule-1. A value of 44.0 x 10(-9) micron 2 molecule-1 was obtained in a single UV-sensitive cone. 5. Substitution of the native dehydroretinal chromophore in the red-sensitive cone pigment with 11-cis-retinal increased the solution photosensitivity to (9.6 +/- 0.62) x 10(-9) micron 2 molecule-1. 6. We conclude that cone pigments have large molecular absorption cross-sections and high quantum efficiencies of photoisomerization. These properties seem well suited for the receptive molecules of a highly sensitive, miniaturized transducer.
View details for Web of Science ID A1991GK91400041
View details for PubMedID 1818565
SYNCHRONOUS BURSTS OF ACTION-POTENTIALS IN GANGLION-CELLS OF THE DEVELOPING MAMMALIAN RETINA
1991; 252 (5008): 939-943
The development of orderly connections in the mammalian visual system depends on action potentials in the optic nerve fibers, even before the retina receives visual input. In particular, it has been suggested that correlated firing of retinal ganglion cells in the same eye directs the segregation of their synaptic terminals into eye-specific layers within the lateral geniculate nucleus. Such correlations in electrical activity were found by simultaneous recording of the extracellular action potentials of up to 100 ganglion cells in the isolated retina of the newborn ferret and the fetal cat. These neurons fired spikes in nearly synchronous bursts lasting a few seconds and separated by 1 to 2 minutes of silence. Individual bursts consisted of a wave of excitation, several hundred micrometers wide, sweeping across the retina at about 100 micrometers per second. These concerted firing patterns have the appropriate spatial and temporal properties to guide the refinement of connections between the retina and the lateral geniculate nucleus.
View details for Web of Science ID A1991FM04000028
View details for PubMedID 2035024
VISUAL TRANSDUCTION IN CONES OF THE MONKEY MACACA-FASCICULARIS
JOURNAL OF PHYSIOLOGY-LONDON
1990; 427: 681-713
1. Visual transduction in macaque cones was studied by measuring the membrane current of single outer segments projecting from small pieces of retina. 2. The response to a brief flash of light was diphasic and resembled the output of a bandpass filter with a peak frequency near 5 Hz. After the initial reduction in dark current there was a rebound increase which resulted from an increase in the number of open light-sensitive channels. The response to a step of light consisted of a prominent initial peak followed by a steady phase of smaller amplitude. 3. Responses to dim light were linear and time-invariant, suggesting that responses to single photons were linearly additive. From the flash sensitivity and the effective collecting area the peak amplitude of the single photon response was estimated as about 30 fA. 4. With flashes of increasing strength the photocurrent amplitude usually saturated along a curve that was gentler than an exponential but steeper than a Michaelis relation. The response reached the half-saturating amplitude at roughly 650 photoisomerizations. 5. The response-intensity relation was flatter in the steady state than shortly after a light step was turned on, indicating that bright light desensitized the transduction with a delay. This desensitization was not due to a reduction in pigment content. In the steady state, a background of intensity I lowered the sensitivity to a weak incremental test flash by a factor 1/(1 + I/IO), where IO was about 2.6 x 10(4) photoisomerizations s-1, or about 3.3 log trolands for the red- and green-sensitive cones. 6. Bleaching exposures produced permanent reductions in flash sensitivity but had little effect on the kinetics or saturating amplitude of subsequent flash responses. The sensitivity reductions were consistent with the expected reductions in visual pigment content and gave photosensitivities of about 8 x 10(-9) microns2 (free solution value) for the red- and green-sensitive pigments. During a steady bleaching exposure the final exponential decline of the photocurrent had a rate constant given by the product of the light intensity and the photosensitivity. 7. In some cells it was possible to measure a light-induced increase in current noise. The power spectrum of the noise resembled the spectrum of the dim flash response and the magnitude of the noise was consistent with a single photon response roughly 20 fA in size. 8. The membrane current recorded in darkness was noisy, with a variance near 0.12 pA2 in the band 0-20 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1990DT90600037
View details for PubMedID 2100987
EFFECTS OF MODIFIED CHROMOPHORES ON THE SPECTRAL SENSITIVITY OF SALAMANDER, SQUIRREL AND MACAQUE CONES
JOURNAL OF PHYSIOLOGY-LONDON
1990; 424: 545-560
1. Chemically modified retinal chromophores were used to investigate the mechanisms that produce the characteristic spectral absorptions of cone pigments. Spectral sensitivities of single cones from the salamander, squirrel and macaque retina were determined by electrical recording. The chromophore was then replaced by bleaching the pigment and regenerating it with a retinal analogue. 2. Exposing a bleached cone to 9-cis-retinal for a brief period (less than 20 min) caused its flash sensitivity to recover to about 0.2 of the pre-bleach value. Similar exposure to a locked 6-s-cis, 9-cis analogue gave a recovery to about 0.03 of the pre-bleach value. 3. Unlike the flash sensitivity, the saturating photocurrent amplitude often recovered completely after bleaching and regenerating the pigment. 4. When the 3-dehydroretinal chromophore in the salamander long-wavelength-sensitive (red) cone was replaced with 11-cis-retinal, shortening the conjugated chain in the chromophore, the spectral sensitivity underwent a blue shift of 67 nm. 5. Pigments containing the planar-locked 6-s-cis.9-cis-retinal analogue absorbed at substantially longer wavelength than those containing unmodified 9-cis-retinal. The opsin shift, a measure of the protein's ability to modify the chromophore's absorption was larger for the locked analogue than for 9-cis-retinal. This suggests that the native chromophore assumes a twisted 6-s-cis conformation in these pigments. 6. The spectral sensitivities of red and green macaque cones containing 9-cis-retinal or planar-locked 6-s-cis.9-cis-retinal retained the 30 nm separation characteristic of the native pigments. This suggests that the different absorptions of of the 6-7 carbon bond in the retinal chromophore.
View details for Web of Science ID A1990DD81900032
View details for PubMedID 2391661
GATING KINETICS OF THE CYCLIC-GMP-ACTIVATED CHANNEL OF RETINAL RODS - FLASH-PHOTOLYSIS AND VOLTAGE-JUMP STUDIES
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (4): 1287-1291
The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester ("caged" cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (greater than 50 microM), whereas at physiological cGMP concentrations (less than 5 microM), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.
View details for Web of Science ID A1988M196200071
View details for PubMedID 2448798
- MOLECULAR MECHANICS OF THE CYCLIC-GMP-ACTIVATED CHANNEL OF RETINAL RODS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 1988; 53: 325-332
SPECTRAL SENSITIVITY OF PRIMATE PHOTORECEPTORS
1988; 1 (3): 255-261
The spectral sensitivities of rods and cones in macaque and human retinas were determined by recording the membrane current from single outer segments. In the macaque retina, the wavelengths of maximum sensitivity were at about 430, 530, and 561 nm for the blue, green, and red cones, respectively, and at 491 nm for the rods. The shapes of the spectra of the three cones were similar when plotted on a log wavenumber scale; the rod spectrum was slightly broader. Spectral sensitivities of the red and green cones from a human retina were virtually identical to those of macaque cones. For comparison with human psychophysical measurements, the rod and cone spectra were adjusted to give the sensitivities expected for light incident on the cornea of the human eye. These functions satisfactorily predicted the scotopic and photopic luminosity functions as well as results from human color-matching experiments. The adjusted spectra of the red and green cones also agreed well with the pi-mechanism of Stiles (1953, 1959).
View details for Web of Science ID A1988Q292300002
View details for PubMedID 3154798
SPECTRAL SENSITIVITY OF CONES OF THE MONKEY MACACA-FASCICULARIS
JOURNAL OF PHYSIOLOGY-LONDON
1987; 390: 145-160
1. Spectral sensitivities of cones in the retina of cynomolgus monkeys were determined by recording photocurrents from single outer segments with a suction electrode. 2. The amplitude and shape of the response to a flash depended upon the number of photons absorbed but not the wave-length, so that the 'Principle of Univariance' was obeyed. 3. Spectra were obtained from five 'blue', twenty 'green', and sixteen 'red' cones. The wave-lengths of maximum sensitivity were approximately 430, 531 and 561 nm, respectively. 4. The spectra of the three types of cones had similar shapes when plotted on a log wave number scale, and were fitted by an empirical expression. 5. There was no evidence for the existence of subclasses of cones with different spectral sensitivities. Within a class, the positions of the individual spectra on the wave-length axis showed a standard deviation of less than 1.5 nm. 6. Psychophysical results on human colour matching (Stiles & Burch, 1955; Stiles & Burch, 1959) were well predicted from the spectral sensitivities of the monkey cones. After correction for pre-retinal absorption and pigment self-screening, the spectra of the red and green cones matched the respective pi 5 and pi 4 mechanisms of Stiles (1953, 1959).
View details for Web of Science ID A1987J654600010
View details for PubMedID 3443931
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