A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.     

The effects of intracellular iontophoretic injection of calcium and sodium ions on the light response of Limulus ventral photoreceptors

During intracellular iontophoretic injection of Ca⁺⁺ into Limulus ventral photoreceptor cells, there is a progressive diminution of the light response. Following Ca⁺⁺ injection, the size of the light response slowly recovers. Similarly, there is a progressive diminution of the light response during intracellular injection of Na⁺ and recovery after the injection is stopped. The rate of diminution during Na⁺ injection is greater for higher [Ca⁺⁺]_out. In solutions which contain 0.1 mM Ca⁺⁺, there is nearly no progressive decrease in the size of the light response during Na⁺ injection. Intracellular injections of Li⁺ or K⁺ do not progressively decrease the size of the light response. We propose that an increase in [Na⁺]_in leads to an increase in [Ca⁺⁺]_in and that an increase in [Ca⁺⁺]_in by any means leads to a reduction in responsiveness to light.     

The initiation of excitation and light adaptation in Limulus ventral photoreceptors

Two types of experiments indicate that light adaptation and excitation are initiated by the same, rather than different, populations of visual pigment. (a) The criterion action spectra of light adaptation and excitation are the same. (b) Increment-threshold curves were measured with a voltage-clamp technique under conditions of high and low concentration of plasma membrane rhodopsin (Rhpm). SD, the dark-adapted sensitivity, and 1/I2, the inverse of the background irradiance that desensitized by 0.3 log units, underwent the same fractional change when the rhodopsin concentration was changed. Both quantities appear to be linearly related to Rhpm. Reversible reductions in Rhpm were achieved by orange irradiation during a brief increase of extracellular pH from 7.8 to 10. This procedure would be unlikely to produce similar concentration changes in a hypothetical intracellular pigment because the concurrent change in intracellular pH, measured using the dye, phenol red, was only 0.45 pH units. It is thus unlikely that an intracellular pigment initiates light adaptation. On the assumption that light adaptation is mediated by a light-induced release of Ca++ from an intracellular store. the results reported here imply that an intracellular transmitter is needed to couple Rhpm to the intracellular store.     

A quantitative comparison of the time-course of sensitivity changes produced by calcium injection and light adaptation in Limulus ventral photoreceptors

The time-course of light and dark adaptation was quantitatively compared with the time-course of the onset of and recovery from desensitization produced by intracellular calcium injection in Limulus ventral photoreceptors. The onset of light adaptation tended to be faster (by 60-90 s) than the onset of desensitization produced by intracellular Ca++ injection. The initial portion of the time-course of dark adaptation was faster (about 10-20 s) than the time-course of recovery from desensitization produced by intracellular Ca++ injection. The final portion of recovery from Ca++ injection had the same time-course as a comparable dark adaptation.     

Local adaptation in the ventral photoreceptors of Limulus

Local adaptation was demonstrated in the ventral photoreceptors of Lumulus using either flashes or continuous illumination. Spots of light locally desensitized the region of the photoreceptor on which they were focused. In dark-adapted photoreceptors where "quantum bumps" were clearly discernible, local adaptation of the quantum bumps was observed. Local adaptation could induce differences of threshold of 1 decade over distances of 50-80 mum. With continuous local illumination these gradients could be maintained from 2 s to 30 min. In addition, the decrease in time scale associated with light adaptation was also found to be localized to the region of illumination.     

Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors

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 approximately 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.     

Excitation and adaptation of Limulus ventral photoreceptors by inositol 1,4,5 triphosphate result from a rise in intracellular calcium

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.     

Ion channels activated by light in Limulus ventral photoreceptors

The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors.     

Central connections of Limulus ventral photoreceptors revealed by intracellular staining

We stained the central terminations of Limulus ventral photoreceptors by intracellular injection of cobalt chloride into the cell bodies. Axons of these photoreceptors enter the protocerebrum via the ventral optic nerve and pass to the medulla. As they reach the surface of the medullar neuropil they branch profusely in fine processes with intermittent varicosities. Each axonal arborization covers about 0.01-0.02 mm2 of this surface immediately adjacent to the medullar ganglion cell layer. Each point on the surface of the medullar neuropil receives, on the average, input from about 6 ventral photoreceptor axons.     

Effects of removing extracellular Ca2+ on excitation and adaptation in Limulus ventral photoreceptors.

In Limulus ventral photoreceptors, removing extracellular calcium (Ca2+o) increases the median latency of light-evoked discrete waves. Removal greatly lengthens the time-to-peak of responses in the dark-adapted cell, but not in the light-adapted cell. Removal does not block light-adaptation or the light-induced rise in intracellular calcium (Ca2+i). These results are interpreted in terms of the hypothesis that both sensitivity and the kinetics of excitation are dependent on Ca2+i, and that Ca2+i is dependent on Ca2+o in the dark-adapted cell, but in the light is dependent largely on Ca2+ released from intracellular compartments.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

Quantitative pressure injection of picoliter volumes into Limulus ventral photoreceptors.

The conformation of the 14 amino acid peptide hormone somatostatin in aqueous solution was investigated through a proton magnetic resonance (PMR) scalar coupling analysis. Experiments were performed at two fields, 270 and 600 MHz, and included double and triple resonance difference scalar decoupling, resolution enhancement and computer simulation. The agreement between simulated and observed spectra at both fields provided support for the correctness of the analysis. The resultant scalar coupling constants, 3J alpha H-NH and 3J alpha B, gave information on the backbone (phi) and side chain (chi 1) torsional angles, respectively, which eliminated either of the proposed conformations of somatostatin as describing a predominant conformer of the molecule in solution under our conditions.     

Calcium ion,a putative intracellular messenger for light-adaptation in Limulus ventral photoreceptors

Calcium ion fulfills several criteria for identifying an intracellular messenger for light-adaptation in Limulus photoreceptors. Direct injection of Ca⁺⁺ mimicks two aspects of light-adaptation; sequestration of intracellular calcium tends to prevent light-adaptation; and light induces an increase in intracellular Ca⁺⁺ as demonstrated by two independent techniques.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Voltage-sensitive potassium channels in Limulus ventral photoreceptors

The steady-state slope conductance of Limulus ventral photoreceptors increases markedly when the membrane is depolarized from rest. The ionic basis of this rectification has been examined with a voltage-clamp technique. Tail currents that occur when membrane potential is repolarized after having been depolarized have been identified. The tail currents reverse direction at a voltage that becomes more positive when Ko is increased. Rectification is reduced by extracellular 4-aminopyridine and by intracellular injection of tetra-ethyl-ammonium (TEA). These results indicate that the membrane rectification around resting potential is due primarily to voltage-sensitive K+ channels. The increase in gK caused by depolarization is not mediated by a voltage-dependent rise in in Cai++, since intracellular injection of Ca++ causes a decrease rather than an increase in slope conductance. TEA can be used to examine the functional role of the K+ channels because it blocks them without substantially affecting the light-activated Na+ conductance. The effect of TEA on response-intensity curves shows that the K+ channels serve to compress the voltage range of receptor potentials.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor.

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A 1 M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, 35SO4 was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3-7.2, to intracellular concentrations of at least 40-200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4-8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Kinetic properties of single-ion channels activated by light in Limulus ventral nerve photoreceptors

Previous results on Limulus ventral photoreceptors have suggested that besides inositol trisphosphate, another unknown transmitter may also work in the transduction cascade. This assumption has been supported by the finding of two light-activated channel types. The present report furnishes further evidence of the dual transmitter mechanism in phototransduction by analyzing the kinetic properties and voltage dependency of these cation channels with conductances of 12 pS and 30 pS. Single-channel currents were recorded in Limulus ventral nerve photoreceptors in cell-attached configuration at 14°C. At V _m + 80 mV the open-time histograms of both channels were fit best by the sum of two exponentials; time constants (and weights) were: 0.81 ms (0.62) and 6.20 ms (0.38) for the 12 pS channels and 2.38 ms (0.43) and 19.4 ms (0.57) for the 30 pS channels. At this potential the mean open times were 2.7 ms for the 12 pS and 13.3 ms for the 30 pS channels, about two-times larger than at hyperpolarizing potentials. The deactivation kinetics were also different for the two channels. The time constants of the decay of the channel activity, after switching off the light, were 2.5 s for the 12 pS and 12.9 s for the 30 pS channels. The 12 pS channel exhibits bursting and subconductance states at positive potentials. The subconductances are about 20%, 46% and 72% of the fully open state. Results show that the two types of light-activated channels have different kinetic parameters, voltage dependence and gating mechanisms. The two channels are suggested to be gated by different transmitters or processes. It is proposed that for the 30 pS channel the transmitter could be calcium ion or a calcium-dependent transmitter.     

Dependence of bump rate and bump size in Limulus ventral nerve photoreceptor on light adaptation and calcium concentration

Bumps were recorded in Limulus ventral nerve photoreceptor as deflections in membrane voltage during 10 s illuminations by dim light which were repeated every 20 s. The bump amplitude vs frequency distribution and its dependence on the intensity of a preadapting light flash are described. Light adaptation which diminishes the average bump amplitude alters the character of the bump amplitude distribution from a curve with a convex region to a continuously falling concave curve. Weak light adaptation can increase frequency (and height) of the bumps elicited by constant stimuli. Raising the external Ca²⁺-concentration from 10 to 40 mmol/l augments the effect of a preadapting light flash in diminishing the bump amplitudes and also increases the bump frequency. The results are consistent with the assumptions

that light adaptation is based on a Ca²⁺-dependent reduction of the amplification factor which determines the bump size and

that the coupling between light induced rhodopsin reactions and bump generation is Ca²⁺-dependent.

     

Light-induced changes of sensitivity in Limulus ventral photoreceptors

The responses of Limulus ventral photoreceptors to brief test flashes and to longer adapting lights were measured under voltage clamp conditions. When the cell was dark adapted, there was a range of energy of the test flashes over which the peak amplitude of the responses (light-induced currents) was directly proportional to the flash energy. This was also true when test flashes were superposed on adapting stimuli but the proportionality constant (termed peak currently/photon) was reduced. The peak current/photon was attenuated more by brighter adapting stimuli than by less bright adapting stimuli. The peak current/photon is a measure of the sensitivity of the conductance-increase mechanism underlying the light response of the photo-receptor. The response elicited by an adapting stimulus had a large initial transient which declined to a smaller plateau. The peak current/photon decreased sharply during the declining phase of the transient and was relatively stable during the plateau. This indicates that the onset of light adaptation is delayed with respect to the onset of the response to the adapting stimulus. If the adaptational state just before the onset of each of a series of adapting stimuli was constant, the amplitude of the transient was a nearly linear function of intensity. When the total intensity was rapidly doubled (or halved) during a plateau response, the total current approximately doubled (or halved). We argue that the transition from transient to plateau, light-elicited changes of threshold, and the nonlinear function relating the plateau response to stimulus intensity all reflect changes of the responsiveness of the conductance-increase mechanism.     

Calcium ion, a putative intracellular messenger for light-adaptation in Limulus ventral photoreceptors.

Calcium ion fulfills several criteria for identifying an intracellular messenger for light-adaptation in Limulus photoreceptors. Direct injection of Ca++ mimicks two aspects of light-adaptation; sequestration of intracellular calcium tends to prevent light-adaptation; and light induces an increase in intracellular Ca++ as demonstrated by two independent techniques.