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     

Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors

Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr).     

Mechanisms regulating variability of the single photon responses of mammalian rod photoreceptors

Variability in the single photon responses of rod photoreceptors limits the accuracy with which the number and timing of photon absorptions are encoded. We investigated how much single photon responses of mammalian rods fluctuate and what mechanisms control these fluctuations. Mammalian rods, like those of toads, generated responses to single photons with trial-to-trial fluctuations 3-4 times smaller than other familiar signals produced by single molecules. We used the properties of the measured fluctuations to constrain models for how the single photon responses are regulated. Neither feedback control of rhodopsin's activity nor saturation within the transduction cascade were consistent with experiment. The measured responses, however, could be explained by multistep shutoff of rhodopsin or a combination of multistep shutoff and saturation.     

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.     

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-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.     

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.     

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.     

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.     

Facilitation of the responses to injections of inositol 1,4,5-trisphosphate analogs in Limulus ventral photoreceptors.

Injection of inositol 1,4,5-trisphosphate and its metabolically resistant analogs InsP3S3 and L-chiro-2,3,5-InsP3 into the ventral photoreceptors of Limulus results in the release of calcium from internal stores and in a current flow into the cells. We show here that the dependence of the current response on the amount of analog injected is supralinear. The injections also facilitate the responses to subsequent injections. We analyze the kinetics of the responses either by very slow application of the analogs directly into the lobe that is sensitive to InsP3 and light or by delivering a pulse into the nonsensitive lobe of the cell, in both cases creating a ramp of rising concentration in the sensitive region. Typically, a long latent period was followed by a strong brief inward current. The ratio between the latency and the duration of the response, defined as twice the time from half-amplitude to the peak of the response, reaches values greater than 10. Our analysis shows that this value cannot be attained within realistic models whose only nonlinearity is the cooperative binding of the ligand to its receptor. The observed ratio, however, can be achieved with a positive feedback model. Treatments that lead to partial depletion of calcium stores reversibly increase the latency of the response. We conclude that the mechanisms of the response of Limulus ventral eye to the metabolically resistant analogs of InsP3 probably involves a positive feedback mechanism and that the carrier of the feedback is likely to be Ca2+.     

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.     

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.     

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.     

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     

Limulus ventral photoreceptors contain a homologue of the alpha-subunit of mammalian Ns

Membranes from ventral photoreceptors of Limulus were incubated with cholera toxin and [32P]NAD+. Cholera toxin catalyzes a specific ADP-ribosylation of a 43-kDa peptide from Limulus ventral photoreceptors. Possible homologies between the 43-kDa peptide of Limulus and the alpha-subunits of mammalian stimulatory, guanine nucleotide-binding regulatory component of adenylate cyclase (Ns) were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of these peptides that are radiolabeled by [32P]NAD+ and cholera toxin. Evidence is provided for structural homology between this invertebrate peptide and mammalian Ns.     

Fast desensitization of the response to InsP3 in Limulus ventral photoreceptors.

In Limulus ventral photoreceptor cells the time-course of the desensitization of InsP3 response was measured by an injection-pair paradigm. Pressure pulses of InsP3 were delivered into the cell with various interpulse intervals. The desensitization of the response to the second injection of each pair approached totality at 200 ms, which is the duration of the response to a single pressure pulse of InsP3. Lowering extracellular calcium did not affect the time-course of the desensitization. Lowering the temperature slowed down both the time-course of the response to InsP3 and the time-course of the desensitization to the same extent. These findings suggest that the desensitization is powerful enough and its onset fast enough to contribute to the transience of the InsP3 response. The time-course of the desensitization suggests it may influence light adaptation.     

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.     

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.     

Localization of the photocurrent of Limulus ventral photoreceptors using a vibrating probe.

We have used a vibrating probe to determine the profile of electrical current density around ventral photoreceptors of the horseshoe crab following flashes of light that uniformly illuminated the entire surface of the photoreceptor's cell body. The vibrating probe signal indicated that the density of inward current was greatest at the distal region of the cell, the region that is expected to contain the light-sensitive rhabdom. The density of inward current typically declined at the midpoint of the cell body and then reversed to an outward current flow in the proximal region of the cell body, close to the axon. The profile of local sensitivity of the photoreceptor to light closely matched the profile of inward current density, suggesting that the light-activated conductance is localized to the light-sensitive region of the cell.