Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid-binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of alpha-tocopherol to either liposomes or an ethanolic-buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9-cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.

Single pressure injections of 1-10 pl of inositol 1,4,5 triphosphate (IP3) or inositol 4,5 bisphosphate [I(4,5)P2] excite Limulus ventral photoreceptors by inducing rapid bursts of inward current. After excitation by IP3, responses to subsequent injections of IP3 or light flashes are often reversibly diminished (adapted). Single injections of IP3 and I(4,5)P2 are effective at concentrations in the injecting pipette of 20 microM to 1 mM. Single injections of inositol 1,4 bisphosphate are ineffective at concentrations of 100-500 microM. Excitation by IP3 or I(4,5)P2 is accompanied by a rise in intracellular free calcium, as indicated by aequorin luminescence. Prior injection of calcium buffer solutions containing 100 mM EGTA greatly diminishes the total charge transferred across the plasma membrane during excitation by IP3 or I(4,5)P2, which suggests that a rise in Cai is necessary for excitation by the inositol polyphosphates. Adaptation of the response to light by IP3 is also abolished by prior injection of EGTA. In the same cells, the response to brief light flashes is slowed and diminished in amplitude by the injection of calcium buffer, but the charge transferred during the response is not significantly diminished. This suggests that light has access to a pathway of excitation in the presence of EGTA that is not accessible to intracellularly injected IP3.

Single pressure injections of 1-2 mM calcium aspartate into the light-sensitive region of Limulus ventral photoreceptors resulted in a rapid, 20-40-mV depolarization lasting -2 s . The depolarization closely followed the rise in intracellular free calcium caused by the injection, as indicated by aequorin luminescence . The depolarization was followed by reversible desensitization (adaptation) of responses to both light and inositol 1,4,5 trisphosphate . Similar single injections of calcium into the light-insensitive region of the receptor were essentially without effect, even though aequorin luminescence indicated a large, rapid rise in intracellular free calcium . The depolarization caused by injection of calcium arose from the activation of an inward current with rectification characteristics and a reversal potential between +10 and +20 mV that were similar to those of the light-activated conductance, which suggests that the same channels were activated by light and by calcium . The reversal potentials of the light-and calcium-activated currents shifted similarly when three-fourths of the extracellular sodium was replaced by sucrose, but were not affected by a similar replacement of sodium by lithium . The current activated by calcium was abolished by prior injection of a calcium buffer solution containing EGTA . The responses of the same cells to brief light flashes were slowed and diminished in amplitude, but were not abolished after the injection of calcium buffer . Light adaptation and prior injection of calcium diminished the calcium-activated current much less than they diminished the light-activated current .

No abstract

Many pharmacological probes must be applied to the interior of cells to produce their effects. Ideally, a method for injecting such materials should be simple, rapid, and independent of the chemical properties of the material to be injected. In addition, one might desire to confirm immediately that an injection occurred and to estimate the volume injected shortly thereafter. We report that these conditions are fulfilled when the injection of materials from micropipettes by pressure pulses is confirmed by visualization of injection-induced disturbances in cells viewed on a video monitor. Volumes of aqueous droplets subsequently injected into a nearby oil pool may be used to estimate the volumes injected into cells. We have obtained a calibration curve for these quantitative estimates of injected volumes by injecting radioactively labeled sulfate into Limulus photoreceptor cells. We find that the estimates are accurate within a range covering one order of magnitude. We assess the sources of systematic and random errors in making these estimates.

The 9-methyl group of 11-s-retinal plays a crucial role in photoexcitation of the visual pigment rhodopsin. A hydrogen-substituted analogue, 11-cis-9-desmethylretlnal, combines with opsin to form a pigment that produces abnormal photoproducts and diinished activation of the GTP-binding protein transducin in vitro. We have measured the formation of this analogue pigment in bleached salamander rods and determined the size and shape of its quantal response. In addition, we have characterized the influence of opsin and newly formed analogue pigment on the quantal response to native porphyropsin. We find that, as 11-cis-9-desmethylretinal combines with opsin in bleached rods, the amplitude of the quantal response from residual native pigment is elevated by 74.5-fold to 0.15 ± 0.09 pA, a value close to the amplitude of the quantal response before bleach (0.31 ± 0.10 pA). When activated by light, the new analogue pigment produces a quantal response that is ==30-fold smaller and decays ""5 times more slowly than that ofnative pigment in unbleached cells. We conclude that the 9-methyl group of retinal is not critical for conversion of opsin to its nondesensitizing state but that it is critical for the normal processes of activation and deactivation of metarhodopsin that give rise to the quantal response.Photoisomerization of li-cis-retinal (Fig. 1, structure 1) initiates an intramolecular rearrangement of rhodopsin that results in a catalytically active state of rhodopsin, R * (1-5). Deactivation of R* requires phosphorylation by rhodopsin kinase (6, 7) and the subsequent binding of arrestin (3,(7)(8)(9). In isolated photoreceptors, pigment activation and deactivation produce a discrete electrical response with a characteristic amplitude and time course (10,11). In an examination of the steric interactions between the apoprotein opsin and its chromophore, Ganter et al. (12) reported that 11-cis-9-desmethylretinal ( Fig. 1, structure 3) produced abnormal photoproducts and transducin activation that was 8% of the rhodopsin control. Here we examine the influence of the 9-methyl group of retinal on the amplitude and shape of the quantal response in isolated rods.To provide access to the ligand binding pocket ofopsin, the native chromophore (13) 11-cis-3,4-dehydroretinal (Fig. 1, structure 2) was removed by bleaching. Bleaching reduces the sensitivity of a cell by depleting the supply of native pigment and by reducing the amplitude of the quantal response from the residual pigment (14)(15)(16)(17). In the absence of li-cis-retinal, this desensitization persists indefinitely (15,18) and is unresponsive to the addition of all-trans-retinal (15,19) or its removal from opsin by hydroxylamine (20,21). We refer to the persistent component of desensitization that results from response attenuation and does not require the presence of a retinal-containing photoproduct as opsin desensitization. Taken together with the loss of sensitivity resulting from pigment depletion, the total loss of sensitivity is commonly referred to as ...

Discrete voltage fluctuations that occur spontaneously or in response to dim lights can be recorded from the ventral photoreceptors of Limulus. The injection of vanadate or the hydrolysis-resistant analog of guanosine triphosphate, GTP-gamma-S, into ventral photoreceptors induces the production of discrete waves in the dark. The chemically induced discrete waves are similar to those induced by light. Ventral photoreceptors may contain a guanyl nucleotide binding protein whose activation by vanadate or GTP-gamma-S induces the discrete waves.