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.     

Calcium mediates the light-induced decrease in maintained K+ current in Limulus ventral photoreceptors

In addition to increasing the conductance to sodium, light reduces the maintained voltage-dependent potassium current (iK) in Limulus ventral photoreceptors. We have investigated the mechanism underlying this long-lasting decrease in ik. Intracellular injection of calcium produced a similar reduction of the voltage-dependent outward current. This reduction was not due to an activation of the voltage-dependent inward current (iin) because calcium injection reduced the outward current even under conditions where iin was blocked with Ni2+, and because calcium injection produced a decrease in conductance, as measured from the slope of the instantaneous i-V curve. The effect of light on ik could be blocked by injection of the calcium buffer EGTA (pCa 7.1) to an intracellular concentration of 50-70 mM. Even larger injections of the pH buffer MOPS (100-200 mM) did not reduce the effect of light on ik. These experiments show that intracellular free calcium (Cai2+) can reduce ik. Furthermore, since Cai2+ is known to increase in light, our results are consistent with the hypothesis that calcium is the internal transmitter for the light-induced decrease in ik.     

The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors

The identity of the transmitter(s) involved in visual transduction in invertebrate microvillar photoreceptors remains unresolved. In this study, the role of inositol 1,4,5-trisphosphate (IP3) was examined in Limulus ventral photoreceptors by studying the effects on the light response of heparin and neomycin, agents that inhibit the production or action of IP3. Both heparin and neomycin reduce responses to brief flashes of light and the transient component of responses to steps of light, and also inhibit IP3-induced calcium release, indicating that IP3 plays a direct role in invertebrate visual excitation. The effects of BAPTA, a calcium buffer, were also examined and shown to be consistent with a role for IP3-mediated calcium release in visual excitation. However, all three agents fail to block the plateau component of the response to a step of light, indicating that a single pathway involving IP3 and calcium cannot solely be responsible for visual excitation in invertebrates. We suggest that the inositol phosphate cascade and a second parallel process that is not dependent on IP3 are involved in the production of the light response.     

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.     

Role of intracellular calcium and sodium in light adaptation in the retina of the honey bee drone (Apis mellifera, L)

In the honey bee drone, the decrease in sensitivity to light of a retinula cell exposed to background illumination was found to be accurately reflected by the difference in amplitude between the initial transient depolarization and the lowest steady depolarization evoked by the background light. It is shown that both the decrease in sensitivity to light and the accompanying drop in potential from the transient to the plateau can be prevented by injecting EGTA intracellularly. A decrease in duration and amplitude of responses to short test flashes such as observed immediately after illumination was found to occur too when Ca or Na, but not K, Li, or Mg injected into dark-adapted retinula cells. Injection of EGTA into a retinula cell maintained a steady state of light adaptation, was found to cause an increase in amplitude and duration of the response to a short test flash, thus producing the effects of dark adaptation. It is suggested that, in the retina of the honey bee drone, an increase in intracellular calcium concentration plays a central role in light adaptation and that an increase in intracellular sodium concentration, resulting from the influx of sodium ions during the responses to light, could lead to this increase in intracellular free calcium.     

Inositol 1,4,5-trisphosphate-induced calcium release is necessary for generating the entire light response of limulus ventral photoreceptors

The experiments reported here were designed to answer the question of whether inositol 1,4,5-trisphosphate (IP3)-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. For this purpose the membrane-permeable IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2APB) (Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. J. Biochem. (Tokyo). 122:498-505) was used. Previously, 2APB was found to inhibit the light activated current of Limulus ventral photoreceptors and reversibly inhibit both light and IP3 induced calcium release as well as the current activated by pressure injection of calcium into the light sensitive lobe of the photoreceptor (Wang, Y., M. Deshpande, and R. Payne. 2002. Cell Calcium. 32:209). In this study 2APB was found to inhibit the response to a flash of light at all light intensities and to inhibit the entire light response to a step of light, that is, both the initial transient and the steady-state components of the response to a step of light were inhibited. The light response in cells injected with the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was reversibly inhibited by 2APB, indicating that these light responses result from IP3-mediated calcium release giving rise to an increase in Cai. The light response obtained from cells after treatment with 100 microM cyclopiazonic acid (CPA), which acts to empty intracellular calcium stores, was reversibly inhibited by 2APB, indicating that the light response after CPA treatment results from IP3-mediated calcium release and a consequent rise in Cai. Together these findings imply that IP3-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors.     

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.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Calcium is necessary for light excitation in barnacle photoreceptors

Summary Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca²⁺ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La³⁺), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca²⁺ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca²⁺, but not K⁺ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La³⁺. The EGTA experiments suggest that internal Ca²⁺ is necessary for light excitation of barnacle photoreceptors while the La³⁺ experiments suggest that La³⁺-sensitive inward current is necessary to maintain excitation during prolonged light.Abbreviations EGTA ethylenglyol-bis-(-aminoethylether) N, N, N¹, N¹-tetraacetate - BAPTA bis-(0-aminophenoxy)-ethane-N, N, N¹, N¹-tetraacetic acid - DMSO dimethyl sulfoxide - trp transient receptor potential - nss no steady state - ASW artificial sea water     

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.     

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.     

Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes

The possible role of Ca ions in mediating the drop in sensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Cai); the latter was measured by using Ca-selective microelectrodes. In dark-adapted photoreceptors, the mean resting Cai was 3.5 +/- 2.5 microM SD (n = 31). No correlation was found between resting Cai and absolute sensitivity from cell to cell. Typically, photoreceptors are not uniformly sensitive to light; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it. In response to a brief flash, the Cai rise was barely detectable when 10(2) photons were absorbed, and it was saturated when approximately 10(5) photons were absorbed. During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity: a 100-fold desensitization was associated with a 2.5-fold increase in Cai. After a bright flash, sensitivity and Cai recovered with different time courses: the cell was still desensitized by approximately 0.5 log units when Cai had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation. Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Cai, which suggests that the time to peak is affected by a change in the capacity to bind Ca2+ and not by resting Cai. Lowering the extracellular Ca2+ concentration (Cao) first decreased Cai and increased sensitivity. Longer exposure to low Cao resulted in a further decrease of Cai but decreased rather than increased sensitivity, which suggests that under certain conditions it is possible to uncouple Cai and sensitivity.     

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors causes an elevation of intracellular free Ca concentration (Cai) and depolarizes the photoreceptors. When measured with the photoprotein aequorin, the InsP3-induced Cai increase follows the time course of depolarization and declines within 1-2 s. However, sensitivity to further injections of InsP3 remains suppressed for several tens of seconds. The possibility that the suppression of Ca release (feedback inhibition) is due to a small lingering elevation of Cai, below the existing detection limit of aequorin, was investigated by measuring Cai with Ca-sensitive electrodes. Double-barreled, Ca- selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Light or InsP3 injections into the light- sensitive compartment depolarized the photoreceptors and induced an elevation of Cai that persisted for tens of seconds. Injections of InsP3 during the decay of Cai showed that sensitivity to InsP3 recovered as resting Cai approached the prestimulus level. The relationship between elevated Cai and feedback inhibition was very steep. An elevation of Cai of 1 microM or more was associated with inhibitions of 79 +/- 12.4% (SEM; n = 7) for the InsP3-induced Cai increase and of 76 +/- 8% for depolarizations. With a residual Cai elevation of 0.01 microM or less, the mean inhibition was 10 +/- 7.4% for InsP3-induced Cai increase and 6.6 +/- 4% for InsP3-induced depolarization. Injections of InsP3 into a light-insensitive compartment within the cell induced elevations of Cai with no associated depolarizations or feedback inhibition. To verify that a sustained elevation of Cai is necessary for inhibition of InsP3-induced Cai increase and depolarization, we injected ethyleneglycol-bis-(beta- aminoethylether)-N,N'-tetraacetic acid (EGTA) between two injections of InsP3. Injection of 1 mM EGTA or the related Ca chelator BAPTA, delivered 750 ms after the first injection of InsP3, restored the peak depolarization caused by the second injection of InsP3 to > 80 +/- 3% of control, compared with 13 +/- 8% without an intervening injection of EGTA. Measurement of Cai with aequorin showed that an intervening injection of EGTA partially restored the InsP3-induced Cai increase. The results suggest that feedback inhibition of InsP3-induced Cai increase and depolarization is mediated by a lingering elevation of Cai and not by depletion of intracellular Ca stores.     

The phosphoinositide signaling cascade is involved in photoreception in the leech Hirudo medicinalis.

The effects of BAPTA, heparin, and neomycin on electrical light responses were studied in the photoreceptors of Hirudo medicinalis. Light activation produces a fast increase in intracellular Ca2+ concentration (Cai) as detected with the fluorescent Ca2+ indicator calcium green-5N. Chelating intracellular calcium by injections of 10 mmol(-1) BAPTA suppresses spontaneous quantum bumps, reduces light sensitivity by more than 2 log(10) units, and substantially increases the latent period of light responses. BAPTA strongly inhibits the plateau phase of responses to long steps of light. Injections of 45-100 mg ml(-1) of heparin act in a similar manner to BAPTA, affecting the latency of the light responses even more. De-N-sulfated heparin, an inactive analog, is almost ineffective at the same concentration compared with heparin. Heparin diminishes the light-induced Cai elevation significantly, whereas de-N-sulfated heparin does not. Intracellular injections of 50-100 mmol l(-1) of the aminoglycoside neomycin, which inhibits phospholipase-C-mediated inositol 1,4,5-trisphosphate formation, acts similar to BAPTA and heparin. Pressure injections of the hydrolysis resistant analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate, strongly depolarize leech photoreceptors and mimic an effect of light adaptation. These results suggest a close similarity between phototransduction mechanisms in leech photoreceptors and existing models for visual transduction in other invertebrate microvillar photoreceptors.     

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.     

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.     

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.