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.     

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.     

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.     

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.     

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.     

2-Aminoethoxydiphenyl borate inhibits phototransduction and blocks voltage-gated potassium channels in Limulus ventral photoreceptors

2-Aminoethoxydiphenyl borate (2-APB) is a membrane-permeable modulator that inhibits the activation of inositol (1,4,5) trisphosphate (InsP(3)) receptors, store operated channels (SOCs) and TRP channels in cells that utilize the phosphoinositide cascade for cellular signaling. In Limulus ventral photoreceptors, light-induced calcium release via the phosphoinositide cascade is thought to activate the photocurrent. Injection of either exogenous InsP(3) or calcium ions can therefore mimic excitation by light. One hundred micromolar 2-APB reversibly inhibited the photocurrent of ventral photoreceptors in a concentration-dependent manner, acting on at least two processes thought to mediate the visual cascade. 2-APB reversibly inhibited both light and InsP(3)-induced calcium release, consistent with its role as an inhibitor of the InsP(3) receptor. In addition, 2-APB reversibly inhibited the activation of depolarizing current flow through the plasma membrane caused by pulsed pressure injection of calcium ions into the light-sensitive lobe of the photoreceptor. We also found that 100 micro M 2-APB reversibly inhibited both transient and sustained voltage-activated potassium current during depolarizing steps. 2-APB has previously been shown to block phototransduction in Drosophila photoreceptors. The lack of specificity of the action of 2-APB in Limulus indicates that this blockade need not necessarily arise from inhibition of InsP(3)-induced calcium release.     

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.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

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.     

Light-induced Mn2+ influx in Limulus ventral photoreceptors

In contrast to insect species, light-activated influx of divalent ions into Limulus ventral photoreceptors has proven difficult to demonstrate. We used the quench of the fluorescent indicator dye, fura-2, to measure Mn²⁺ influx. Limulus ventral photoreceptors were injected with fura-2 and excited at 360 nm. When the photoreceptors were bathed in 1 mmol · l⁻¹ Mn²⁺, an approximately 1% per 10 s decline in the fura-2 fluorescence during intervals between 50-ms flashes was taken as a measure of Mn²⁺ entry in darkness. Fluorescence decline during 10-s flashes was used to monitor Mn²⁺ entry during the photoresponse. During the 10-s flashes we observed a small rapid decline of the fura-2 fluorescence even in the absence of Mn²⁺. This reflected a contamination of the fluorescence signal arising from light-induced release of intracellular calcium stores. A subsequent slower decline in fluorescence during the 10-s flash, amounting to approximately 9% per 10 s, was only observed in the presence of extracellular Mn²⁺ and was attributed to Mn²⁺ influx. This light-activated influx was not through voltage-gated calcium channels since it persisted under voltage clamp, was not stimulated by depolarizing current injections, nor blocked by NiCl₂. Depletion of internal calcium stores by cyclopiazonic acid treatment did not accelerate Mn²⁺ influx. Accepted: 30 January 1998     

Flash photolysis of caged compounds in Limulus ventral photoreceptors

Rapid concentration jumps of Ins(1,4,5)P3 or ATP were made inside Limulus ventral photoreceptors by flash photolysis of the parent caged compounds. In intact ventral photoreceptors, the photolysis flash evokes a maximum amplitude light-activated current; therefore, a procedure was developed for uncoupling phototransduction by blocking two of the initial reactions in the cascade, rhodopsin excitation and G protein activation. Rhodopsin was inactivated by exposure to hydroxylamine and bright light. This procedure abolished the early receptor potential and reduced the quantum efficiency by 325 +/- 90-fold (mean +/- SD). G protein activation was blocked by injection of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). GDP beta S injection reduced the quantum efficiency by 1,881 +/- 1,153-fold (mean +/- SD). Together hydroxylamine exposure and GDP beta S injection reduced the quantum efficiency by 870,000 +/- 650,000-fold (mean +/- SD). After the combined treatment, photoreceptors produced quantum bumps to light that was approximately 10(6) times brighter than the intensity that produced quantum bumps before treatment. Experiments were performed with caged compounds injected into photoreceptors in which phototransduction was largely uncoupled. Photolysis of one compound, myo-inositol 1,4,5-triphosphate P4(5)-1-(2-nitrophenyl)ethyl ester (caged IP3), increased the voltage clamp current in response to the flashlamp by more than twofold without changing the latency of the response. The effect was not seen with photolysis of either adenosine-5'-triphosphate P3-1-(2-nitrophenyl)ethyl ester (caged ATP) or caged IP3 in cells preloaded with either heparin or (1,2-bis-(o-amino-phenoxy)ethane-N-N-N'-N' tetraacetic acid tetrapotassium salt (BAPTA). The results suggest that photoreleased IP3 releases calcium ions from intracellular stores and the resulting increase in [Ca2+]i enhances the amplification of the phototransduction cascade.     

Functional significance of voltage-dependent conductances in Limulus ventral photoreceptors

The influence of voltage-dependent conductances on the receptor potential of Limulus ventral photoreceptors was investigated. During prolonged, bright illumination, the receptor potential consists of an initial transient phase followed by a smaller plateau phase. Generally, a spike appears on the rising edge of the transient phase, and often a dip occurs between the transient and plateau. Block of the rapidly inactivating outward current, iA, by 4-aminopyridine eliminates the dip under some conditions. Block of maintained outward current by internal tetraethylammonium increases the height of the plateau phase, but does not eliminate the dip. Block of the voltage-dependent Na+ and Ca2+ current by external Ni2+ eliminates the spike. The voltage-dependent Ca2+ conductance also influences the sensitivity of the photoreceptor to light as indicated by the following evidence: depolarizing voltage- clamp pulses reduce sensitivity to light. This reduction is blocked by removal of external Ca2+ or by block of inward Ca2+ current with Ni2+. The reduction of sensitivity depends on the amplitude of the pulse, reaching a maximum at or approximately +15 mV. The voltage dependence is consistent with the hypothesis that the desensitization results from passive Ca2+ entry through a voltage-dependent conductance.     

Adapting bump model for ventral photoreceptors of Limulus

Light-evoked current fluctuations have been recorded from ventral photoreceptors of Limulus for light intensity from threshold up to 10(5) times threshold. These data are analyzed in terms of the adapting bump noise model, which postulates that (a) the response to light is a summation of bumps; and (b) the average size of bump decreases with light intensity, and this is the major mechanism of light adaptation. It is shown here that this model can account for the data well. Furthermore, the model provides a convenient framework to characterize, in terms of bump parameters, the effects of calcium ions, which are known to affect photoreceptor functions. From responses to very dim light, it is found that the average impulse response (average of a large number of responses to dim flashes) can be predicted from knowledge of both the noise characteristics under steady light and the dispersion of latencies of individual bumps. Over the range of light intensities studied, it is shown that (a) the bump rate increases in strict proportionality to light intensity, up to approximately 10(5) bumps per second; and (b) the bump height decreases approximately as the -0.7 power of light intensity; at rates greater than 10(5) bumps per second, the conductance change associated with the single bump seems to reach a minimum value of approximately 10(-11) reciprocal ohms; (c) from the lowest to the highest light intensity, the bump duration decreases approximately by a factor of 2, and the time scale of the dispersion of latencies of individual bumps decreases approximately by a factor of 3; (d) removal of calcium ions from the bath lengthens the latency process and causes an increase in bump height but appears to have no effect on either the bump rate or the bump duration.     

Light-activated single channel currents in Limulus ventral nerve photoreceptors

Light-activated single channel currents were measured in Limulus ventral photoreceptors in the cell-attached configuration at 14°C. The results show three channel types with conductances of 6.2, 10.4 and 28.7 pS. The most active channels have the 10 pS conductance; the open time histograms of these channels could be best fitted by the sum of two exponentials with time constants (and weights) of 0.58 ms (0.78) and 4.32 ms (0.22), suggesting two populations of channels or two open states. The mean open time was 1.38 ms. The open time histogram of the channels with the 29 pS conductance could be best fitted by a single exponential with a time constant of 3.35 ms. First latencies of the 10 pS channels were between 40 and 280 ms but those of the 29 pS conductance channels were 300 ms. These findings suggest that the two channel types are gated by two different intracellular transmitters or mechanisms. Offprint requests to: K. Nagy     

Light reduces the voltage-dependent inward current in Limulus ventral photoreceptors

In Limulus ventral photoreceptors, illumination not only increases a specialized light-activated sodium conductance but also modulates voltage-dependent conductances. Previous work has demonstrated that the delayed rectifier current is reduced by light; we report here that the early voltage-dependent inward current is also reduced by light. Furthermore, by maintained during continuous depolarization and that this maintained inward current can be reduced by light. EGTA injection was found to increase the maintained inward current.     

Subcellular localization of neutral red staining in Limulus ventral photoreceptors

Limulus ventral photoreceptors are vitally stained by neutral red. In other systems such staining has been correlated with the presence of monoamines or neuropeptides. The stained cellular components in ventral photoreceptors are clusters of small ovoids which have been identified as residual bodies. These structures are unlikely candidates for monoamine or neuropeptide synthesis or storage sites, but may be part of the cyclic synthesis and degradation of photosensitive membrane. While vital staining with neutral red is a particularly useful method for identifying certain classes of neurons in vivo, in the case of ventral photoreceptors, the association of the vital staining property with the presence of a particular class of neurotransmitter candidates has proven difficult. Neutral red is useful, however, for visualizing the segmentation of ventral photoreceptors in vivo.     

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.     

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.     

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.