Limulus Ventral Photoreceptors Research Articles

Testing the role of calmodulin in the excitation of Limulus photoreceptors

The phototransduction cascade in Limulus ventral photoreceptors involves multiple second messengers, including Ca 2+ and cGMP. Light-induced Ca 2+ release from intracellular stores is an intermediate step, but the subsequent Ca 2+-activated reaction remains to be determined. The possibility that Ca 2+/calmodulin (Ca 2+/CaM) might be involved is suggested by the high calmodulin content of the transducing lobe. To test whether CaM can excite the transduction cascade we injected a 25 μM Ca 2+/CaM solution. This produced a rapid, brief depolarization similar to that produced by light, suggesting a role for CaM in the cascade. However, an important caveat is that Ca 2+ dissociating from the Ca 2+/CaM complex might excite this process. Several control experiments argue against, but do not entirely eliminate this possibility. To test whether endogenous CaM has a function in excitation, trifluoperazine was pressure injected into the rhabdomeric region. The response to brief flashes was not affected, but the response to steady illumination was transiently attenuated by each injection. We conclude that calmodulin should be considered a candidate to couple intermediate and late stages of the transduction cascade.

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+ release and the opening of cGMP-gated channels

BackgroundEarly stages in the excitation cascade of Limulus photoreceptors are mediated by activation of Gq by rhodopsin, generation of inositol-1,4,5-trisphosphate by phospholipase-C and the release of Ca2+. At the end of the cascade, cGMP-gated channels open and generate the depolarizing receptor potential. A major unresolved issue is the intermediate process by which Ca2+ elevation leads to channel opening.ResultsTo explore the role of guanylate cyclase (GC) as a potential intermediate, we used the GC inhibitor guanosine 5'-tetraphosphate (GtetP). Its specificity in vivo was supported by its ability to reduce the depolarization produced by the phosphodiesterase inhibitor IBMX. To determine if GC acts subsequent to InsP3 production in the cascade, we examined the effect of intracellular injection of GtetP on the excitation caused by InsP3 injection. This form of excitation and the response to light were both greatly reduced by GtetP, and they recovered in parallel. Similarly, GtetP reduced the excitation caused by intracellular injection of Ca2+. In contrast, this GC inhibitor did not affect the excitation produced by injection of a cGMP analog.ConclusionWe conclude that GC is downstream of InsP3-induced Ca2+ release and is the final enzymatic step of the excitation cascade. This is the first invertebrate rhabdomeric photoreceptor for which transduction can be traced from rhodopsin photoisomerization to ion channel opening.

Desensitization of the photoresponse in Limulus ventral photoreceptors by protein kinase C precedes rhabdomere disorganization and endocytosis.

Limulus photoreceptors utilize the phosphoinositide pathway to generate light-induced single photon events (quantum bumps) that sum to form the depolarizing receptor potential. The protein kinase C (PKC) activator, (-)-indolactam V (ILV) rapidly desensitizes the light response in Limulus ventral nerve photoreceptors. Within 10 min of extracellular application, 100 nM (-)-ILV caused a decrease in the mean amplitude of quantum bumps to 38% of control values. PKC activation by (-)-ILV also causes photosensitive membrane disorganization and endocytosis. To investigate whether this precedes desensitization of the electrical response, we fixed cells after 10-min incubation with 25 microM (-)-ILV, a concentration sufficient to cause a 1000-fold desensitization of the receptor potential. The photosensitive microvilli of these photoreceptors remained narrow, densely packed, and well organized. Increasing the incubation time to 60 min did, however, induce disorganization and swelling of the microvilli and endocytosis of the photosensitive membrane, as previously reported. Measurement of membrane capacitance did not indicate a significant reduction in membrane area accompanying desensitization by (-)-ILV. PKC-induced reduction in light sensitivity therefore precedes the detection of ultrastructural changes in the rhabdomeral membrane and is not due to a net loss of membrane.

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 μM 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.

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 μM 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.

Simultaneous roles for Ca2+ in excitation and adaptation of Limulus ventral photoreceptors.

The ventral photoreceptors of Limulus have been one of the main preparations for the study of invertebrate phototransduction. The study of ventral photoreceptors has revealed that they have remarkable performance characteristics, most notably the very large amplification of the transduction process. This amplification is critically dependent upon the coupling of photoactivated rhodopsin to the phosphoinositide cascade, resulting in the release of Ca2+ from intracellular stores. The consequent elevation of Ca2+ within the photoreceptor's cytosol is amongst the most rapid and dramatic known to be activated by the phosphoinositide cascade. This review summarizes the evidence that intracellular Ca2+ is a key regulator of transduction in Limulus photoreceptors. The mechanisms that regulate Ca2+ as well as the possible targets of the action of Ca2+ are reviewed. Ca2+ elevation is critical for triggering both excitation and adaptation processes in the photoreceptor. The question of how a single second messenger can produce these two opposing effects is of obvious interest and is a topic dealt with throughout this review.

Protein kinase C‐induced disorganization and endocytosis of photosensitive membrane in Limulus ventral photoreceptors

Protein kinase C (PKC) desensitizes the light response in photoreceptors from the ventral optic nerve of the horseshoe crab Limulus. Photoisomerization of Limulus rhodopsin leads to phosphoinositide hydrolysis, resulting in the production of inositol trisphosphate and diacylglycerol (DAG). Inositol trisphosphate mobilizes intracellular stores of Ca(2+), resulting in photoreceptor excitation in Limulus, while DAG may activate PKC. We investigated whether PKC-mediated desensitization of the photoresponse is accompanied by ultrastructural changes in the rhodopsin-bearing photosensitive membrane (rhabdom) in Limulus ventral photoreceptors. PKC activation by (-)-indolactam V in darkness induces disorganization and swelling of the rhodopsin-containing microvilli and endocytosis of rhabdomeral membrane. The effects of (-)-indolactam V on dark-adapted photoreceptor ultrastructure are reversible, are stereospecific, are blocked by coapplication of PKC inhibitors, and closely match those induced by continuous, bright light. Rhabdom disorganization and endocytosis via PKC activation may, therefore, contribute to desensitization of the light-adapted photoreceptor.

Divergent mechanisms for phototransduction of invertebrate microvillar photoreceptors.

Recently, it was reported that the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA) and linolenic acid (LNA), activate the light-sensitive channels in Drosophila photoreceptors (Chyb et al. 1999). We have examined whether these PUFAs activate the light-sensitive channels in Limulus ventral photoreceptors. We find that, whether applied from the outside or injected into a Limulus ventral photoreceptor, either AA or LNA fails to activate the light-sensitive channels. Moreover, the synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, also fails to activate the light-sensitive channels. We suggest that these findings require us to rethink our view about the generality of the process of phototransduction in invertebrate microvillar photoreceptors. We propose that the photoreceptors of Drosophila and Limulus evolved to utilize different branches of the phosphoinositide pathway for phototransduction: those of Limulus evolved to utilize IP3-mediated calcium release while those of Drosophila evolved to utilize diacylglycerol and it's downstream products.

Timing of Ca(2+) release from intracellular stores and the electrical response of Limulus ventral photoreceptors to dim flashes.

Light-induced release of Ca(2+) from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca(2+) indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 microm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca(i)) after flashes delivering between 10 and 5 x 10(5) effective photons. Peak Ca(i) increased with flash intensity to 138 +/- 76 microM after flashes delivering approximately 10(4) effective photons, while the latent period of the elevation of Ca(i) fell from approximately 140 to 21 ms. The onset of the light-induced elevation of Ca(i) was always highly correlated with that of the receptor potential. The time for Ca(i) to exceed 2 microM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 +/- 6.4 ms). Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca(i) and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca(2+) ions that appears necessary for a highly amplified electrical response to dim flashes.

Effects of Calcium and Cyclopiazonic Acid on the Photoresponse in the Limulus Ventral Photoreceptor

Abstract 1. Single-photon responses (bumps) and small macroscopic photocurrents were studied in ventral photoreceptors of the horseshoe crab Limulus. Lowering the calcium concentration in the bath from 10 mᴍ to 250 μᴍ led to increased bump size. Adaptation of the cells by a moderately bright conditioning flash was not impaired. 2. Pressure-injection of 1.2 mᴍ EGTA into the dark-adapted cells resulted in reduced bump size. EGTA weakened the effect of the conditioning light flash although it did not completely abolish light adaptation. 3. The microsomal calcium-ATPase inhibitor cyclopiazonic acid strongly desensitized the cells, and bumps were suppressed below detection. When the bathing saline contained 10 mᴍ calcium, macroscopic photoresponses after extracellular application of the agent had amplitudes smaller than under control conditions but normal response kinetics: The response to a light step still consisted of a fast transient photocurrent and a much smaller plateau. However, when applied in calcium-free bathing saline, cyclopiazonic acid additionally influenced the waveform of the photoresponse. The clear distinction between transient and plateau was no longer possible, and the photocurrent appeared “square”. 4. Our results support the idea that a transient elevation of the cytosolic calcium concentration is obligatory for light adaptation in the ventral photoreceptor. It is also obligatory for the generation of the so-called C2 component of the photocurrent which is represented by “standard” bumps and the fast transient phase of a prolonged response. However, a rise in cytosolic calcium appears not necessary for the initiation of a slow electrical photoresponse.

Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus.

Cyclic nucleotide-gated channels have been proposed to mediate the electrical response to light in the ventral photoreceptor cells of the horseshoe crab, Limulus polyphemus. However, a cyclic nucleotide-gated channel has not been identified from Limulus. We have cloned a putative full-length cyclic nucleotide-gated channel cDNA by screening cDNA libraries constructed from Limulus brain using a probe developed from Limulus ventral eye nerves. The putative full-length cDNA was derived from two overlapping partial cDNA clones. The open reading frame encodes 905 amino acids; the sequence shows 44% identity to that of the alpha subunit of the bovine rod cyclic GMP-gated channel over the region containing the transmembrane domains and the cyclic nucleotide binding domain. This Limulus channel has a novel C-terminal region of approximately 200 amino acids, containing three putative Src homology domain 3 binding motifs and a putative coiled-coil domain. The possibility that this cloned channel is the same as that detected previously in excised patches from the photoreceptive membrane of Limulus ventral photoreceptors is discussed in terms of its sequence and its expression in the ventral eye nerves.

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.

A role for hydrolysis of inositol 1,4,5-trisphosphate in terminating the response to inositol 1,4,5-trisphosphate and to a flash of light in Limulus ventral photoreceptors

Injection of inositol 1,4,5-trisphosphate (Ins 1,4,5-P 3) into Limulus ventral photoreceptors produces excitation similar to that produced by light. One process which might contribute to rapid termination of the responses to Ins 1,4,5-P 3 and to light is the hydrolysis of Ins 1,4,5-P 3 by an InsP 3-5-phosphatase to form inositol 1,4-bisphosphate. Inositol 2,4,5-trisphosphate (Ins 2,4,5-P 3) is known to be less hydrolysable by the InsP 3-5-phosphatase than is Ins 1,4,5-P 3. Whereas ventral photoreceptors respond to an injection of Ins 1,4,5-P 3 with a single wave of depolarization, the response to Ins 2,4,5-P 3 is a burst of waves of depolarization. Our hypothesis is that it is the resistance to hydrolysis by the InsP 3-5-phosphatase which accounts for the burst of waves produced by Ins 2,4,5-P 3. To test this idea we injected ventral photoreceptors with Ins 1,4,5-P 3 in the presence of the non-specific phosphatase inhibitors, vanadate and fluoride, which prolong the response to a flash of light in ventral photoreceptors (D.W. Corson, A. Fein, W.W. Walthall, J. Gen. Physiol. 82 (1983) 659–677). In the presence of fluoride or vanadate the response to Ins 1,4,5-P 3 was composed of a burst of waves rather than a single wave of depolarization. We conclude that hydrolysis of Ins 1,4,5-P 3 by the InsP 3-5-phosphatase plays a role in terminating the ventral photoreceptors response to Ins 1,4,5-P 3 and also to light.

Cytosolic Calcium Transient and Light Response in Limulus Ventral Nerve Photoreceptors: Supralinearity in Response-Intensity Curves

Abstract Light-evoked intracellular electrical responses were measured simultaneously with the changes in intracellular free Ca2+ or Sr2+ concentration by means of arsenazo III. The response-versus-stimulus intensity characteristics was measured in two states of relative light adaptation in strontium-containing saline (10 mᴍ Sr2+; 0.25 mᴍ Ca2+). Supralinear parts of the curves with slopes > 1 were found for the receptor current amplitude and the current­-time integral as well as for the amplitude of the arsenazo signal within at least 0.3 log units of identical stimulus intensity range. Maximum slopes of response-versus-intensity curves are on average approximately 1.7 for the receptor current and the arsenazo signal in both adaptational states. The rise in time of the arsenazo signal evoked by a light flash shows two phases. In strontium saline we observe a fast increase at the onset of the arsenazo signal which is detectable within the first 20-100 ms of the signal rise and a slow further increase to the maximum, with a duration of 0.7-6 s. In the detailed analysis of both phases we observed that the amplitude of each phase increases supralinear, i.e. disproportionate to the stimulus intensity. The fast rising phase of the arsenazo signal is not visibly voltage-dependent. This fast rising phase might be a monitor of an IP3-sensitive calcium or strontium release. Since the amplitude of the fast rising phase increases supralinear with the stimulus intensity, we propose the interpretation that more than one IP3-molecule must bind to the IP3-receptor to open this channel of the intracellular calcium store. Only the slow rising phase is voltage-dependent. The kinetic of this slow rising phase was accelerated with more negative membrane potentials. This predominant slow phase correlates with the receptor current-time integral. Therefore the slow rising phase of the arsenazo signal might be a monitor for influx of calcium or strontium through light activated ion channels in the plasma membrane. The supralinearity of the amplitude of the slow rising phase might indicate the supralinear increase in calcium or strontium, which is carried by the receptor current. Additionally, we calculated the fraction of strontium and calcium ions of the total receptor current. When calcium was replaced by strontium in the superfusate about 3-30% of the receptor current are carried by strontium and calcium ions. In physiological saline calcium contributes about 1-1.5% to the receptor current.

Limulus ventral photoreceptors contain two functionally dissimilar inositol triphosphate-induced calcium release mechanisms

Injections of inositol 1,4,5-triphosphate (InsP 3) into Limulus ventral photoreceptors give rise to a rapid depolarization and an elevation of intracellular calcium concentration (Ca i). This response to InsP 3 is followed by a period of desensitization that persists as long as Ca i remains elevated (feedback inhibition). Limulus ventral photoreceptors have two types of lobe: a light-sensitive rhabdomeric lobe (R lobe), and a light-insensitive arhabdomeric lobe (A lobe). Evidence showing the presence of feedback inhibition has been so far demonstrated only in the R lobe. In this study, simultaneous measurements of Ca i were made using aequorin and double-barreled calcium-sensitive electrodes in each type of lobe. We carefully checked the location of the R lobe and A lobe by scanning a microspot of light across the whole photoreceptor. We then inserted a double-barreled calcium-sensitive microelectrode with InsP 3 in either type of lobe. In the R lobe, injections of InsP 3 led to a large Ca i increase, a rapid depolarization and feedback inhibition; a brief flash of light induced a rapid depolarization and a Ca i increase measured by both aequorin and the calcium-sensitive electrode. In the A lobe, injection of InsP 3 led to an increase in Ca i measured by the calcium-sensitive electrode but to no depolarization or aequorin luminescence. Further there was no evidence of feedback inhibition in the A lobe; the elevation of Ca i caused by the first injection did not desensitize the photoreceptor to a second injection of InsP 3 3 s later. To verify that the aequorin and the cell membrane respond to an increase in Ca i, we presented a brief flash of light. Following a uniform illumination, there is indeed a typical large luminescence increase and a receptor potential. The calcium-sensitive electrode measures a small and slow Ca i increase because its tip is located in the A lobe and it is measuring Ca 2+ diffusing from the R lobe. Our observation that the InsP 3-induced Ca i increase in the A lobe is not apparently accompanied by a subsequent desensitization to InsP 3 may suggest that there are more than one type of InsP 3 receptor in the same cell. Alternatively, the InsP 3 receptor could be the same but some additional factor, which confers feedback inhibition, could be missing in the A lobe.

Latencies of calcium elevation and depolarization in Limulus ventral photoreceptors injected with GDP-βS

We have used confocal fluorescence microscopy and fluorescent calcium indicators to investigate the relationship between light-induced elevation of intracellular calcium ion concentration (Ca i) and depolarization in small volumes of cytosol close to the microvillar plasma membrane of the ventral photoreceptor of Limulus polyphemus. We prolonged the latency of the light response by treatment of cells with hydroxylamine and injection of the G-protein blocker, guanosine-5′-O-(2-thiodiphosphate (GDP-βS). Such treatment increased the latency of the cell's response from approximately 20 to 50 ms. In both treated and untreated cells we observed a close correlation between the times at which we first detected the electrical response and the elevation of Ca i. We obtained 18 out of 54 and 12 out of 22 recordings, in untreated and treated cells respectively, for which the elevation of Ca i was detected simultaneous with, or 1–4 ms prior to, the electrical response to light. The prolonged latent period exhibited by treated cells may make possible future investigation of the effects on the initial response to light of local photolytic release of caged compounds at the microvillar membrane.

Differential calcium sensitivity of two types of quantum bumps in Limulus ventral nerve photoreceptors

Ventral photoreceptors in Limulus polyphemus were continuously illuminated with dim light and produced two types of quantum bumps. Bumps of one type had a relatively symmetrical shape and fast rise and decay kinetics. In addition, bumps were detected with a small amplitude and slower kinetics. We termed these bump types “C2” and “C1 bumps”, respectively. The half width of a bump divided by its amplitude was found to be a reliable criterion to distinguish between the bump types. This shape quotient is smaller than 0.5 ms/pA for the C2 bumps and larger than 1 ms/pA for the C1 bumps. Lowering the extracellular calcium concentration from 10 mM to 0.25 mM caused an increase in the average amplitude of C2 bumps to 196%. After the injection of small amounts of 1.2 mM EGTA solution the amplitude of these bumps was reduced to about 30 and 50% in two cells studied. By contrast, in both cases C1 bump amplitudes were not affected significantly. We conclude that the two bump types are generated in one photoreceptor by the activation of different transduction pathways. These pathways are differentially sensitive to changes of the cytosolic calcium concentration.

P-G1-07 Use of caged compounds and microphotolysis to study phototransduction in Limulus ventral photoreceptors : Ukhanov K, Payne R. Dept. Zool., Univ. of Maryland, College Park, MD 20742, USA

P-G1-07 Use of caged compounds and microphotolysis to study phototransduction in Limulus ventral photoreceptors : Ukhanov K, Payne R. Dept. Zool., Univ. of Maryland, College Park, MD 20742, USA

Current components stimulated by different G-proteins in Limulus ventral photoreceptor.

The components of the light-activated current in ventral photoreceptor of Limulus have different physical and pharmacological properties, indicating the operation of different transduction pathways. We investigated whether these pathways are activated by different G-proteins. An antiserum, directed against the C-terminal sequence QLNLKEYNLV of the alpha subunit of the Gq-protein, which is present in squid photoreceptor, was injected into the ventral photoreceptor of Limulus. This Gq-alpha mediates the light-dependent activation of PLC. Injections of the antibody into the photoreceptor blocked the second component of the light-evoked current in Limulus ventral photoreceptor, but the other components could still be activated. The results support our earlier hypothesis that the transduction pathways of the current components are selectively activated by different G-proteins.

Light activated calcium release in Limulus ventral photoreceptors as revealed by laser confocal microscopy

Using confocal imaging and fluorescent calcium indicators, light-induced elevation of intracellular Ca 2+ concentration ([Ca 2+]i) in Limulus ventral photoreceptors was shown to be initiated within 4 μm of the light-sensitive plasma membrane. Within 500 ms, elevation of [Ca 2+]i spread throughout the light-sensitive rhabdomeral lobe of the photoreceptor, but barely penetrated the arhabdomeral lobe. During saturating illumination of measurement spots near the plasma membrane, [Ca 2+]i rose at rates of 1–2 mM/s after a latent period of 14–40 ms, reaching peak concentrations of ∼150 μM. Rapid elevation of [Ca 2+]i persisted in the absence of extracellular Ca 2+ and was therefore ascribed to release from intracellular stores. The elevation of [Ca 2+]i was always detectable within 5 ms of the electrical response of the photoreceptor to light. In 14 out of 54 measurements, detection of elevated calcium preceded the electrical response. Cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca-pumps, greatly reduced the elevation of [Ca 2+]i during bright flashes and the sensitivity of the electrical response to dim flashes. However, the maximal response to bright flashes was not diminished. Therefore, although the calcium release that we detect may be fast enough to contribute to the electrical response we are unable to demonstrate that it is absolutely required.