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

The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between... Background: Early stages in the excitation cascade of Limulus photoreceptors are mediated by activation of G by rhodopsin, generation of inositol-1,4,5-trisphosphate by phospholipase-C and 2+ the release of Ca . 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 2+ Ca elevation leads to channel opening. Results: To 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 InsP production in the cascade, we examined the effect of intracellular injection of GtetP on the excitation caused by InsP injection. This form of excitation and the response to light were both greatly reduced by GtetP, and they recovered in parallel. Similarly, 2+ GtetP reduced the excitation caused by intracellular injection of Ca . In contrast, this GC inhibitor did not affect the excitation produced by injection of a cGMP analog. Conclusion: We 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. K selective conductance [2]. In invertebrate rhabdomeric Background Phototransduction processes in invertebrates have both photoreceptors, which also depolarize in response to similarities and differences from that in vertebrate rods. light, no complete transduction cascade has been deter- The initial enzymatic step in all photoreceptors is the acti- mined. It is clear that G protein activates phospholipase C vation of G protein by rhodopsin. In the ciliary photore- in all cases examined so far, including Drosophila [3-5], ceptors of vertebrate rods and cones, G protein activates Limulus [6,7] and squid [8,9]. PLC then hydrolyzes phos- phosphodiesterase leading to a decrease of cGMP concen- phatidylinositol-4,5-bisphosphate to produce inositol- tration, closure of cyclic nucleotide-gated channels and 1,4,5-trisphosphate and diacylglycerol. membrane hyperpolarization (for review see [1]). On the other hand, the ciliary photoreceptors from scallops, Subsequent steps differ among these photoreceptors. In hyperpolarize due to an increase in cGMP which opens a late stages of the excitation cascade in Drosophila, Page 1 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 diacylglycerol (or metabolites) may lead to channel open- quickly than with other antagonists [27]. GtetP was ing [10,11]. However, understanding the final stages has injected until it decreased the light response by at least been hampered by the unavailability of a direct assay for 80%. IBMX was then reapplied. Under these conditions, the light-dependent channels and varying results using the peak depolarization caused by IBMX of 11 mV was heterologous expression systems [12]. In the photorecep- 54% smaller compared to what occurred before GtetP tors of Limulus ventral eye (for review see [13]), the cas- injection (Fig. 1A, GtetP). The maximum slope of the 2+ cade involves PLC, InsP , Ca and cGMP. Light produces depolarization also decreased: during control perfusion of 2+ an InsP -induced Ca elevation that precedes the onset of IBMX, the maximum was 13.6 mV/min, and after injec- the receptor potential [14]. Furthermore, intracellular tions the maximum slope was 6.1 mV/min. In ten experi- 2+ injection of Ca mimics the light response [15-17] and ments, the average decrease of depolarization was 56 ± 2+ buffering intracellular Ca inhibits it [16,18]. Taken 24% (Fig. 1B) and the average decrease in the maximal ris- 2+ together, these results establish that InsP -mediated Ca ing slope was 60 ± 20% (Fig. 1C). These results are con- elevation is an integral part of the excitation cascade. The sistent with GtetP inhibiting GC, thereby opposing the Limulus cascade ends with the opening of cGMP-gated increase in cGMP resulting from PDE inhibition. channels which, in this system, can be directly studied in 2+ cell-attached and excised patches [19,20]. Photoreceptor GC inhibitors act downstream from InsP mediated Ca cells contain mRNA for a putative Limulus cyclic nucle- release 2+ otide-gated channel protein, and antibodies to the In order to provide a link between light-induced Ca ele- expressed protein specifically label the light-sensitive vation and the opening of cGMP-dependent channels, GC 2+ rhabdomeric lobe [21,22]. Furthermore either intracellu- activity must be downstream from Ca in the signaling lar injection of cGMP [23,24] or elevation of cGMP by cascade. To determine if this is the case, photoreceptors 2+ inhibition of phosphodiesterase [25,26] excites the cell. were excited by injecting InsP or Ca directly into the There is thus little doubt that the end of the cascade light-transducing lobe (the R-lobe) [6,7,15-17]. If GC is involves cGMP-gated channels. What remains unclear is downstream, this form of excitation should be reduced by 2+ the mechanism that couples Ca release to cGMP GC inhibitors. A similar strategy has been used previously elevation. to characterize the ordering of other steps in the cascade [15,18,28,29]. Recent work demonstrated that inhibitors of guanylate cyclase strongly reduce the response to light [27]. We first tested whether a GC inhibitor affects the excita- Although these results support the requirement for cGMP tion produced by activating InsP receptors (Fig. 2). Cells during excitation, they do not indicate at which stage GC were impaled with two microelectrodes. One microelec- is involved. In this paper, we test the hypothesis that GC trode contained the poorly hydrolysable analog of InsP , is a missing link in the cascade; i.e. that it acts downstream 3dInsP (1 mM) [30] and was inserted into the R-lobe. 2+ 2+ from Ca elevation as required if cGMP is to couple Ca Previous work has shown that brief injections of InsP or elevation to channel opening. Our results indicate that its analogs excite ventral photoreceptors and that the this is indeed the case. Because PDE inactivation is latency of the response is lowest when the injection bolus unlikely to be involved in excitation (see Discussion), it is close to the light-transducing membrane in the R-lobe appears that activation of GC is what elevates cGMP. It is [6,7]. The second electrode contained 50 mM GtetP and therefore now possible to a give a rather complete picture was positioned in the non-transducing A-lobe. Since mul- of this complex cascade that couples rhodopsin photoi- tiple injections from this microelectrode were spread over somerization to ion channel opening. time, GtetP could diffuse throughout the cell. Each injec- tion of 3dInsP caused a transient repeatable depolariza- Results tion similar to a light response, as previously reported for Guanylate cyclase antagonists oppose the effects of PDE InsP and analogs [31-33]. Cells were then injected with inhibitors sufficient GtetP to cause a substantial decrease in the light Inhibitors of PDE raise cGMP levels in the Limulus eyes response (81% in Fig. 2). Injections of 3dInsP were inter- [26] and produce a depolarization of the photoreceptor spersed between light flashes. It can be seen (Fig. 2) that membrane [25]. GC inhibitors should counteract this the response to the InsP analog was also greatly reduced effect. To reduce PDE activity, 2.5 mM IBMX was added to (81%). In five experiments, the average inhibition of the the bath for several minutes. Fig. 1A shows that this 3dInsP response was 77 ± 16%, comparable to the aver- evoked a 24 mV membrane depolarization in this cell age inhibition of the light response (89 ± 7%). After ces- (control). Once the cell recovered following wash-out of sation of GtetP injections, there was a slow recovery of IBMX, GC inhibitor was injected. We used the competitive both the response to 3dInsP and the response to light. We GC inhibitor guanosine 5'-tetraphosphate because it can found that limiting the number of 3dInsP injections was be injected with greater ease and effects reverse more important for maintaining a consistent response and so Page 2 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. Control GtetP 10 mV 1 min B. C. 40 - 16- 12- 20 - 8 - + + + + + + + + + + + + 4 - + + + + - 0 - Control GtetP Control GtetP Gu Figure 1 anosine 5'-tetraphosphate decreased and slowed the depolarization produced by 2.5 mM IBMX. Guanosine 5'-tetraphosphate decreased and slowed the depolarization produced by 2.5 mM IBMX. (A) Bath application of 2.5 mM IBMX produced a characteristic depolarization of Limulus photoreceptors (control) that was diminished following intracellular pressure injection sufficient to inhibit the light response from a microelectrode containing 25 mM GtetP (GtetP). (B) Amplitudes of IBMX-induced depolarization in individual photoreceptors are matched before and after inhibition of the light response using GtetP. The thick line indicates the average decrease in depolarization (n = 10). (C) The maximum rising slopes of IBMX-induced depolarization in the same photoreceptors as in (B) are matched before and after inhibition of the light response using GtetP. The thick line indicates the average decrease in rising slope. gave approximately ten injections each under control, rather than Na , and that its effects are downstream from GtetP inhibition, and recovery conditions. The 3dInsP activation of InsP receptors. 3 3 used in these experiments was a hexasodium salt (6 mM + + Na in the injection electrode). Since Na in high concen- Similar experiments were done to test whether GtetP 2+ tration can inhibit the light response [34], control experi- inhibits responses to Ca injections (Fig. 3). The response + 2+ ments were done to test whether comparable small Na to Ca injection was strongly inhibited (Fig. 3A), indicat- 2+ injections might account for the observed effect on the ing that GC is downstream from Ca elevation. The insets 2+ light response. In five experiments, no effect of compara- show averaged responses to Ca injection (Fig. 3A) and ble injections of 5 mM Na alone (not shown) was seen. light (Fig. 3B) before and after GtetP-induced inhibition. 2+ We conclude that the effects of GtetP are due to GtetP The time course of inhibition was similar for Ca Page 3 of 11 (page number not for citation purposes) Depolarization (mV) Maximum Slope (mV/min) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 CONTROL INHIBITED RECOVERY 1 35 LIGHT 5 mV 250 ms 2 4 6 3dInsP 3,4 5 min GtetP Injection 2+ Gu Figure 2 anosine 5'-tetraphosphate acts subsequent to InsP -mediated Ca release during excitation. 2+ Guanosine 5'-tetraphosphate acts subsequent to InsP -mediated Ca release during excitation. Intracellular pressure injection from a microelectrode containing 25 mM GtetP decreased both the responses to a test flash and intracellu- lar pressure injection of 1 mM 3dInsP . Brackets and numbers match sets of five consecutive responses to light (1, 3, 5) or 3dInsP (2, 4, 6) averaged to produce the respective trace in the inset. 3dInsP injections were interspersed between test 3 3 flashes during the periods indicated by brackets (2, 4, 6). GtetP was injected during the period indicated by a solid bar. Aver- aged responses are shown before (1, 2), at the end of drug application (3, 4), and late in recovery (5, 6). responses and light responses, however there was some case, our results clearly show that a major component of 2+ quantitative difference: responses to light were decreased Ca -induced excitation can be blocked by a GC inhibitor. 2+ by 90% whereas responses to Ca were decreased by 60% in this experiment. In six experiments, the average inhibi- GC inhibitors act prior to the opening of cyclic nucleotide tion of the light response was 88 ± 7 % and the average gated channels 2+ inhibition of the response to Ca injection was 60 ± 27 In a final set of experiments, we tested the possibility the %. These small differences have not been analyzed fur- GC inhibitor might directly antagonize cyclic nucleotide- ther. One possibility is that the greater inhibition of the gated channels. We know of no precedent or other reason light response is indicative of a minor effect of GtetP on to suspect that GtetP would affect these channels, but it 2+ excitation upstream of InsP -mediated Ca release. In any was nevertheless important to test directly for this possi- bility. This was done by examining whether GtetP affected Page 4 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. 1 mV 200 ms 0 25 50 75 100 125 GtetP Injections B. 20 mV 200 ms 025 50 75 100 125 Time (min) 2+ Gt Figure 3 etP acts subsequent to Ca -mediated excitation. 2+ GtetP acts subsequent to Ca -mediated excitation. (A) Injection from a microelectrode containing 25 mM GtetP 2+ caused a progressive decline in the response to injection from a second microelectrode of 1.8 mM Ca solution buffered with 2+ 2 mM HEDTA. Data points are the average response with error bars (std. dev.) to ten consecutive Ca injections before, after inhibition of the light response by GtetP, and late in recovery of the light response. GtetP was injected during the period indi- cated by the bar positioned between the graphs. Brackets and arrows match response amplitude to averaged voltage time 2+ course in the insets. (B) GtetP injection inhibited the response to test flashes in parallel to the decline in response to Ca . Page 5 of 11 (page number not for citation purposes) 2+ Light Response (mV) Ca response (mV) + BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. B. 1.5 25 - 1.0 - * 10 - 0.5- 5 - Control GtetP Control GtetP Flash cGMP 5 mV 1 mV 200 ms 200 ms Gte Figure 4 tP acts prior to opening of cyclic nucleotide-gated channels. GtetP acts prior to opening of cyclic nucleotide-gated channels. (A) Injection from a microelectrode containing 25 mM GtetP was used to desensitize cells to a test flash by 90% (left panel). Data points are the average response with error bars (std. dev.) to seven consecutive test flashes. (B) The response to injection from a microelectrode containing 250 uM Rp- 8pCPT-cGMPS (cGMP) in the same three cells was qualitatively unaffected by GtetP (left panel). Respective responses before (control) and after injection (GtetP) are matched by lines and symbols (+, *, and open circles). The voltage traces represent averaged responses from one cell (*) to light (left) and Rp-8pCPT-cGMPS (right) before (control) and after GtetP intracellular injections. the excitation produced by intracellular injection of the for each measurement low (n < 10). In control experi- cGMP analog, Rp-8pCPT-cGMPS. We minimized intracel- ments using these conditions (not shown) the response to lular accumulation of this membrane-permeant, high- Rp-8pCPT-cGMPS, the response to light, and membrane affinity agonist by keeping the number of injections used properties remained stable over long periods. Fig. 4 shows Page 6 of 11 (page number not for citation purposes) Peak Amplitude (mV) Peak Amplitude (mV) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 2+ 2+ that the response to Rp-8pCPT-cGMPS was relatively intracellular Ca [15-17] and thwarted by Ca buffers 2+ unaffected by GtetP (10 to 30% decrease, N = 3), whereas [16,18]. Ca elevation is thus necessary and sufficient for the light response decreased enormously (90%). In two excitation. additional cells, the response to Rp-8pCPT-cGMPS injec- tion appeared qualitatively unaffected by GtetP, but prob- Several lines of work indicate that the final step is the acti- lems with clogging, a tendency of microelectrodes vation of cGMP-gated channels. Excitation can be induced containing Rp-8pCPT-cGMPS, precluded quantitative by PDE inhibitors [25,47] or by intracellular injection of analysis. cGMP [23,24]. Most importantly, cGMP can directly acti- vate channels when applied to inside-out excised mem- Discussion brane patches from the R-lobe [19]. These channels have There has been substantial previous work on the pho- properties similar to the light-activated channels in cell- totransduction cascade in Limulus, but the reactions attached patches on the R-lobe [48]. Most recently, a puta- involved in the late stages of the process have been tive cyclic nucleotide-gated channel gene has been cloned unclear. In particular, there has been no information on from Limulus [22]. The mRNA for the channel is expressed 2+ -mediated Ca enzymatic steps downstream from InsP in photoreceptors and the protein product was specifically elevation that might couple this elevation to channel localized in the R-lobe [21]. opening. Recently, it was shown that GC was required in the cascade [27], but its position in the cascade was not The work reported here shows that GC is appropriately 2+ known. Here we have demonstrated that GC is down- positioned in the cascade to couple the light-induced Ca 2+ stream from Ca and thus situated appropriately to medi- elevation to the production of cGMP. In principle, the role ate late stages of the cascade. As a result, a rather complete of GC could be simply to constitutively produce cGMP; picture of the transduction cascade is now possible. In the during light cGMP might be elevated due to a decrease in paragraphs below, we provide an overview of this cascade PDE activity. However, such a decrease in PDE activity and delineate areas where gaps remain. during light exposure would probably enhance the response to injected cGMP relative to the dark-adapted In Limulus, excitation is initiated by conversion of rho- response and certainly not decrease it, the observed effect dopsin to metarhodopsin by light (Fig. 5). The active state [24]. These results thus strongly suggest that the GC is acti- 2+ of metarhodopsin activates a G protein as evidenced by vated as a result of the light-induced elevation of Ca . the fact that G protein inhibitors decrease the light- Because there are few photoreceptors in the ventral eye, response, whereas G protein activators mimic the light this preparation is not well-suited for biochemistry to the 2+ response [31,35,36]. Metarhodopsin is inactivated in less extent that experiments to test for the Ca or light- than 150 ms [37]; while active about 10 G proteins are dependence of GC are not practical. Therefore what is turned on [38]. The G protein involved has been identi- known in these photoreceptors about GC is based on its fied as G in Limulus [39,40]. In the next stage of the cas- pharmacological profile. It has been concluded that the cade, PLC is activated by G , resulting in the hydrolysis of GC involved is not the soluble, NO-dependent form and 2+ phosphatidyl inositol-4,5-bisphosphate to produce InsP therefore does not rely on Ca -dependent activation of and diacyglycerol. PLC antagonists such as neomycin, nitric oxide synthase [27]. An important unresolved issue 2+ spermine, and U-73122 decrease the response to light is how the enzyme might be regulated by Ca . Several 2+ precedents for Ca -dependent activation of this enzyme [41,42] (see also [15]). Diacyglycerol may be important 2+ for excitation in Drosophila [10]; however in Limulus this is must be considered. For instance, in vertebrates Ca - not likely to be the case [43]. InsP has been shown to dependent GC activating proteins (CD-GCAPs) and neu- meet all the requirements for acting as an intracellular sec- rocalcin are known to activate rod GC [49,50]. The con- 2+ ond messenger necessary for excitation in Limulus: endog- centration of Ca required for this activity is well within enous synthesis, increased concentration in response to the range achieved during Limulus phototransduction light, and excitation through exogenous application [6,7]. [44,45]. In ciliates there is a form of GC that can be acti- 2+ vated by Ca /calmodulin [51]. This raises the question of 2+ InsP produces a Ca efflux from intracellular stores and whether GC activation in Limulus might be mediated by 2+ can raise cytosolic Ca upwards of 150 µ M [6,7,44,45]. calmodulin. The involvement of calmodulin in a critical Excitation by light or InsP is blocked by the InsP receptor step in the transduction cascade could be one reason for 3 3 antagonist heparin [18,29]. Direct measurements show the high concentrations of calmodulin found in Limulus 2+ that Ca release is sufficiently fast to activate the light- R-lobes [52]. dependent conductance [14,45]. The InsP receptor is localized in the endoplasmic reticulum adjacent to the The Limulus cascade is more complex than that of the ver- base of the rhodopsin-containing microvilli at the site of tebrate rod, but this increased complexity can be viewed 2+ Ca release [46]. Excitation can be mimicked by raising in light of the remarkable performance characteristics of Page 7 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 Hυ R Rhodopsin hodopsin Metarhodopsin * Metarhodopsin * G GDP GDP G GTP * GTP * PLC PLC PLC-Gq * PLC-Gq * PIP PIP InsP InsP 2+ 2+ Ca Ca ER ER 2+ 2+ Ca Ca cytoplasm cytoplasm GC GC GC* GC* GTP GTP cGMP cGMP CNGC CNGC closed closed C CNGC NGC open open A m Figure 5 odel for Limulus excitation. A model for Limulus excitation. The cascade is initiated by the isomerization of rhodopsin to metarhodopsin by light. Metarhodopsin catalyzes exchange of GTP for GDP on multiple G proteins (G ). G -GTP binds and activates phospholipase C q q 2+ (PLC). This complex cleaves phosphatidyl inositol-4,5-bisphosphate (PIP ) producing InsP . InsP opens Ca ion channels in the 2 3 3 2+ 2+ endoplasmic reticulum (ER) leading to the release of Ca into the cytosol. Ca release activates GC. A rise in cGMP opens cyclic nucleotide-gated ion channels (CNCG) in the plasma membrane. Page 8 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 Limulus photoreceptors. These cells generate single tion [15]. GtetP, HEDTA, and IBMX were obtained from photon responses in the nA range, three orders of magni- Sigma; InsP and 3dInsP from Calbiochem; Rp-8pCPT- 3 3 tude larger than those of the rod. Furthermore, Limulus cGMPS from Biolog. photoreceptors respond over nearly 4 orders of magni- tude greater range of light intensities than rods [53,54]. Microscopy The Limulus cascade has eight stages compared to the five The selection and observation of cells has been described stages of the rod cascade. The larger number of stages may in detail elsewhere [27]. Briefly, cells were observed under underlie the greater single-photon response and wider infrared illumination with Hofmann optics using a Cooke dynamic range seen in Limulus photoreceptors. Corporation Sensicam. Cells were chosen on the basis of having a stable membrane potential and robust dark Conclusions adapted and single photon light responses. Although much has been determined about the pho- totransduction cascade in Limulus, the late steps occurring In some experiments the electrodes had to be placed into 2+ between InsP -induced Ca elevation and the opening of the light-sensitive R lobe of the cells. Under Hoffman the cGMP-gated channels has been unclear. Previous work optics, the R-lobe has a smooth appearance, contrasted showed that guanylate cyclase was necessary for with the granular appearance of the light-insensitive A- generation of the light-response, but did not identify lobe. In these cases, the tip of the electrode was positioned where in the cascade it acted [27]. The major question at the border of the two lobes and advanced axially into answered in the present study is to determine whether GC the R-lobe. is appropriately positioned at the end of the cascade 2+ where it could couple Ca elevation to cGMP elevation. Intracellular pressure injection Our conclusion is that this is the case; the excitation pro- Injection electrodes were backfilled with at least 2 µL of 2+ duced by either InsP or Ca injection can be greatly solution and routinely recorded membrane potentials reduced by inhibiting GC (Figs. 2, 3). Importantly the GC approximately 20 mV higher than 3 M KCl electrodes. inhibitor did not affect the excitation produced by injec- Injections were observed on a monitor. The pulse dura- tion of cGMP analog (Fig. 4); therefore channel function tion and pressure were adjusted to maintain a constant appears unaffected. Taken together with previous results, bolus size. Injection electrodes became clogged on occa- a picture of the enzymatic steps by which rhodopsin is sion, and the blockage was cleared using either a manually coupled to channel activation in an invertebrate rhabdo- controlled high pressure pulse or brief (< 30 ms) oscilla- meric photoreceptor can now be proposed (Fig. 5). tion train. If the cell's light or drug injection responses were affected by the clearing procedure, that experiment The simplest interpretation of the available data is that GC was not used. activation is the primary means by which the intracellular concentration of cGMP is increased during excitation in Abbreviations Limulus photoreceptors. This hypothesis can be tested by A-lobe arhabdomeric lobe characterizing the specific guanylate cylase involved and 2+ the link between Ca release and cyclase activation. 3dInsP 3-deoxy-D-myo-inositol-1,4,5-trisphosphate Methods GC guanylate cyclase Electrophysiology The dissection and techniques for electrophysiology have GtetP guanosine-5'-tetraphosphate been described in detail elsewhere [16]. Cells were exposed to stimulus light with a maximal light intensity of HEDTA N-(2-hydroxyethyl)ethylenediaminetriacetic acid 1.0 mW/cm which was attenuated by neutral density fil- ND ters (attenuation = 10 ). Cells were perfused with artifi- IBMX 3-isobutyl-1-methylxanthine cial sea water (ASW) with the composition (in mM) 425 NaCl, 10 KCl, 10 CaCl , 22 MgCl , 26 MgSO , and 15 Tris, InsP D-myo-inositol-1,4,5-trisphosphate 2 2 4 3 adjusted to pH 7.8. Non-injecting intracellular microelec- trodes contained 3 M KCl (15–25 mΩ resistance). Injec- PDE phosphodiesterase tion microelectrodes contained drugs as described in the text with (in mM) 150 KCl, 10 HEPES, 0.001% Triton X- PLC phospholipase C 100 [17] and had 7–15 mΩ resistance. The microelec- 2+ 2+ trodes used to inject Ca contained 1.8 mM Ca buffered R-lobe rhabdomeric lobe with HEDTA. This use of HEDTA has been described else- where and shown to not affect excitation or light adapta- Page 9 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 17. 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Baer KM, Saibil HR: Light- and GTP-activated hydrolysis of into Limulus ventral photoreceptors causes oscillations of phosphatidylinositol bisphosphate in squid photoreceptor free cytosolic calcium. J.Gen.Physiol. 1991, 97:1165-1186. membranes. J.Biol.Chem. 1988, 263:17-20. 33. Levitan I, Payne R, Potter BV, Hillman P: Facilitation of the 10. Chyb S, Raghu P, Hardie RC: Polyunsaturated fatty acids acti- responses to injections of inositol 1,4,5- trisphosphate ana- vate the Drosophila light-sensitive channels TRP and TRPL. logs in Limulus ventral photoreceptors. Biophysical Journal 1994, Nature 1999, 397:255-259. 67:1161-1172. 11. Hardie RC, Martin F, Cochrane GW, Juusola M, Georgiev P, Raghu P: 34. Lisman JE, Brown JE: The effects of intracellular iontophoretic Molecular basis of amplification in Drosophila phototrans- injection of calcium and sodium ions on the light response of duction: roles for G protein, phospholipase C, and diacylglyc- Limulus ventral photoreceptors. J.Gen.Physiol. 1972, 59:701-719. erol kinase. Neuron 2002, 36:689-701. 35. Fein A, Corson DW: Excitation of Limulus photoreceptors by 12. Hardie RC: Regulation of TRP channels via lipid second vanadate and by a hydrolysis-resistant analog of guanosine messengers. Annu Rev Physiol 2003, 65:735-759. triphosphate. Science 1981, 212:555-557. 13. Lisman JE, Richard EA, Raghavachari S, Payne R: Simultaneous roles 36. Fein A, Corson DW: Both photons and fluoride ions excite for Ca2+ in excitation and adaptation of Limulus ventral Limulus ventral photoreceptors. Science 1979, 204:77-79. photoreceptors. Adv Exp Med Biol 2002, 514:507-538. 37. Richard EA, Lisman JE: Rhodopsin inactivation is a modulated 14. Payne R, Demas J: Timing of Ca2+ release from intracellular process in Limulus photoreceptors. Nature 1992, 356:336-338. stores and the electrical response of Limulus ventral pho- 38. Kirkwood A, Weiner D, Lisman JE: An estimate of the number of toreceptors to dim flashes. J Gen Physiol 2000, 115:735-748. G regulator proteins activated per excited rhodopsin in liv- 15. Richard EA, Ghosh S, Lowenstein JM, Lisman JE: Ca2+/calmodulin- ing Limulus ventral photoreceptors. Proc.Natl.Acad.Sci.U.S.A. binding peptides block phototransduction in Limulus ventral 1989, 86:3872-3876. photoreceptors: evidence for direct inhibition of phospholi- 39. Dorlöchter M, Klemeit M, Stieve H: Immunological demonstra- pase C. Proc.Natl.Acad.Sci.USA 1997, 94:14095-14099. tion of Gq-protein in Limulus photoreceptors. Vis.Neurosci. 16. Shin J, Richard EA, Lisman JE: Ca2+ is an obligatory intermediate 1997, 14:287-292. in the excitation cascade of Limulus photoreceptors. Neuron 40. Munger SD, Schremser-Berlin J-L, Brink CM, Battelle B-A: Molecular 1993, 11:845-855. and immunological characterization of a Gq protein from ventral and lateral eye of the horseshoe crab Limulus polyphemus. Invert. Neurosci. 1996, 2:175-182. 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Ukhanov K, Ukhanova M, Taylor CW, Payne R: Putative inositol 1,4,5-trisphosphate receptor localized to endoplasmic retic- ulum in Limulus photoreceptors. Neuroscience 1998, 86:23-28. 47. Johnson EC, O'Day PM: Inhibitors of cyclic-GMP phosphodi- esterase alter excitation of Limulus ventral photoreceptors in Ca2+-dependent fashion. J.Neurosci. 1995, 15:6586-6591. 48. Bacigalupo J, Chinn K, Lisman JE: Ion channels activated by light in Limulus ventral photoreceptors. J.Gen.Physiol. 1986, 87:73-89. 49. Margulis A, Pozdnyakov N, Sitaramayya A: Activation of bovine photoreceptor guanylate cyclase by S100 proteins. Biochem Biophys Res Commun 1996, 218:243-247. 50. Kumar VD, Vijay-Kumar S, Krishnan A, Duda T, Sharma RK: A sec- ond calcium regulator of rod outer segment membrane gua- nylate cyclase, ROS-GC1: neurocalcin. Biochemistry 1999, 38:12614-12620. 51. Schultz JE, Klumpp S: Lanthanum dissociates calmodulin from the guanylate cyclase of the excitable ciliary membrane from Paramecium. FEMS Microbiol.Letts. 1982, 13:303-306. 52. Battelle BA, Dabdoub A, Malone MA, Andrews AW, Cacciatore C, Calman BG, Smith WC, Payne R: Immunocytochemical localiza- tion of opsin, visual arrestin, myosin III, and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors. J Comp Neurol 2001, 435:211-225. 53. Lisman JE, Brown JE: Light induced changes of sensitivity in Limulus ventral photoreceptors. J.Gen.Physiol. 1975, 66:473-488. 54. Brown JE, Coles JA: Saturation of the response to light in Limu- lus ventral photoreceptors. Journal of Physiology 1979, 296:373-392. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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The excitation cascade of Limulus ventral photoreceptors: guanylate cyclase as the link between InsP3-mediated Ca2+release and the opening of cGMP-gated channels

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Springer Journals
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Copyright © 2004 by Garger et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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1471-2202
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10.1186/1471-2202-5-7
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15053840
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Abstract

Background: Early stages in the excitation cascade of Limulus photoreceptors are mediated by activation of G by rhodopsin, generation of inositol-1,4,5-trisphosphate by phospholipase-C and 2+ the release of Ca . 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 2+ Ca elevation leads to channel opening. Results: To 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 InsP production in the cascade, we examined the effect of intracellular injection of GtetP on the excitation caused by InsP injection. This form of excitation and the response to light were both greatly reduced by GtetP, and they recovered in parallel. Similarly, 2+ GtetP reduced the excitation caused by intracellular injection of Ca . In contrast, this GC inhibitor did not affect the excitation produced by injection of a cGMP analog. Conclusion: We 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. K selective conductance [2]. In invertebrate rhabdomeric Background Phototransduction processes in invertebrates have both photoreceptors, which also depolarize in response to similarities and differences from that in vertebrate rods. light, no complete transduction cascade has been deter- The initial enzymatic step in all photoreceptors is the acti- mined. It is clear that G protein activates phospholipase C vation of G protein by rhodopsin. In the ciliary photore- in all cases examined so far, including Drosophila [3-5], ceptors of vertebrate rods and cones, G protein activates Limulus [6,7] and squid [8,9]. PLC then hydrolyzes phos- phosphodiesterase leading to a decrease of cGMP concen- phatidylinositol-4,5-bisphosphate to produce inositol- tration, closure of cyclic nucleotide-gated channels and 1,4,5-trisphosphate and diacylglycerol. membrane hyperpolarization (for review see [1]). On the other hand, the ciliary photoreceptors from scallops, Subsequent steps differ among these photoreceptors. In hyperpolarize due to an increase in cGMP which opens a late stages of the excitation cascade in Drosophila, Page 1 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 diacylglycerol (or metabolites) may lead to channel open- quickly than with other antagonists [27]. GtetP was ing [10,11]. However, understanding the final stages has injected until it decreased the light response by at least been hampered by the unavailability of a direct assay for 80%. IBMX was then reapplied. Under these conditions, the light-dependent channels and varying results using the peak depolarization caused by IBMX of 11 mV was heterologous expression systems [12]. In the photorecep- 54% smaller compared to what occurred before GtetP tors of Limulus ventral eye (for review see [13]), the cas- injection (Fig. 1A, GtetP). The maximum slope of the 2+ cade involves PLC, InsP , Ca and cGMP. Light produces depolarization also decreased: during control perfusion of 2+ an InsP -induced Ca elevation that precedes the onset of IBMX, the maximum was 13.6 mV/min, and after injec- the receptor potential [14]. Furthermore, intracellular tions the maximum slope was 6.1 mV/min. In ten experi- 2+ injection of Ca mimics the light response [15-17] and ments, the average decrease of depolarization was 56 ± 2+ buffering intracellular Ca inhibits it [16,18]. Taken 24% (Fig. 1B) and the average decrease in the maximal ris- 2+ together, these results establish that InsP -mediated Ca ing slope was 60 ± 20% (Fig. 1C). These results are con- elevation is an integral part of the excitation cascade. The sistent with GtetP inhibiting GC, thereby opposing the Limulus cascade ends with the opening of cGMP-gated increase in cGMP resulting from PDE inhibition. channels which, in this system, can be directly studied in 2+ cell-attached and excised patches [19,20]. Photoreceptor GC inhibitors act downstream from InsP mediated Ca cells contain mRNA for a putative Limulus cyclic nucle- release 2+ otide-gated channel protein, and antibodies to the In order to provide a link between light-induced Ca ele- expressed protein specifically label the light-sensitive vation and the opening of cGMP-dependent channels, GC 2+ rhabdomeric lobe [21,22]. Furthermore either intracellu- activity must be downstream from Ca in the signaling lar injection of cGMP [23,24] or elevation of cGMP by cascade. To determine if this is the case, photoreceptors 2+ inhibition of phosphodiesterase [25,26] excites the cell. were excited by injecting InsP or Ca directly into the There is thus little doubt that the end of the cascade light-transducing lobe (the R-lobe) [6,7,15-17]. If GC is involves cGMP-gated channels. What remains unclear is downstream, this form of excitation should be reduced by 2+ the mechanism that couples Ca release to cGMP GC inhibitors. A similar strategy has been used previously elevation. to characterize the ordering of other steps in the cascade [15,18,28,29]. Recent work demonstrated that inhibitors of guanylate cyclase strongly reduce the response to light [27]. We first tested whether a GC inhibitor affects the excita- Although these results support the requirement for cGMP tion produced by activating InsP receptors (Fig. 2). Cells during excitation, they do not indicate at which stage GC were impaled with two microelectrodes. One microelec- is involved. In this paper, we test the hypothesis that GC trode contained the poorly hydrolysable analog of InsP , is a missing link in the cascade; i.e. that it acts downstream 3dInsP (1 mM) [30] and was inserted into the R-lobe. 2+ 2+ from Ca elevation as required if cGMP is to couple Ca Previous work has shown that brief injections of InsP or elevation to channel opening. Our results indicate that its analogs excite ventral photoreceptors and that the this is indeed the case. Because PDE inactivation is latency of the response is lowest when the injection bolus unlikely to be involved in excitation (see Discussion), it is close to the light-transducing membrane in the R-lobe appears that activation of GC is what elevates cGMP. It is [6,7]. The second electrode contained 50 mM GtetP and therefore now possible to a give a rather complete picture was positioned in the non-transducing A-lobe. Since mul- of this complex cascade that couples rhodopsin photoi- tiple injections from this microelectrode were spread over somerization to ion channel opening. time, GtetP could diffuse throughout the cell. Each injec- tion of 3dInsP caused a transient repeatable depolariza- Results tion similar to a light response, as previously reported for Guanylate cyclase antagonists oppose the effects of PDE InsP and analogs [31-33]. Cells were then injected with inhibitors sufficient GtetP to cause a substantial decrease in the light Inhibitors of PDE raise cGMP levels in the Limulus eyes response (81% in Fig. 2). Injections of 3dInsP were inter- [26] and produce a depolarization of the photoreceptor spersed between light flashes. It can be seen (Fig. 2) that membrane [25]. GC inhibitors should counteract this the response to the InsP analog was also greatly reduced effect. To reduce PDE activity, 2.5 mM IBMX was added to (81%). In five experiments, the average inhibition of the the bath for several minutes. Fig. 1A shows that this 3dInsP response was 77 ± 16%, comparable to the aver- evoked a 24 mV membrane depolarization in this cell age inhibition of the light response (89 ± 7%). After ces- (control). Once the cell recovered following wash-out of sation of GtetP injections, there was a slow recovery of IBMX, GC inhibitor was injected. We used the competitive both the response to 3dInsP and the response to light. We GC inhibitor guanosine 5'-tetraphosphate because it can found that limiting the number of 3dInsP injections was be injected with greater ease and effects reverse more important for maintaining a consistent response and so Page 2 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. Control GtetP 10 mV 1 min B. C. 40 - 16- 12- 20 - 8 - + + + + + + + + + + + + 4 - + + + + - 0 - Control GtetP Control GtetP Gu Figure 1 anosine 5'-tetraphosphate decreased and slowed the depolarization produced by 2.5 mM IBMX. Guanosine 5'-tetraphosphate decreased and slowed the depolarization produced by 2.5 mM IBMX. (A) Bath application of 2.5 mM IBMX produced a characteristic depolarization of Limulus photoreceptors (control) that was diminished following intracellular pressure injection sufficient to inhibit the light response from a microelectrode containing 25 mM GtetP (GtetP). (B) Amplitudes of IBMX-induced depolarization in individual photoreceptors are matched before and after inhibition of the light response using GtetP. The thick line indicates the average decrease in depolarization (n = 10). (C) The maximum rising slopes of IBMX-induced depolarization in the same photoreceptors as in (B) are matched before and after inhibition of the light response using GtetP. The thick line indicates the average decrease in rising slope. gave approximately ten injections each under control, rather than Na , and that its effects are downstream from GtetP inhibition, and recovery conditions. The 3dInsP activation of InsP receptors. 3 3 used in these experiments was a hexasodium salt (6 mM + + Na in the injection electrode). Since Na in high concen- Similar experiments were done to test whether GtetP 2+ tration can inhibit the light response [34], control experi- inhibits responses to Ca injections (Fig. 3). The response + 2+ ments were done to test whether comparable small Na to Ca injection was strongly inhibited (Fig. 3A), indicat- 2+ injections might account for the observed effect on the ing that GC is downstream from Ca elevation. The insets 2+ light response. In five experiments, no effect of compara- show averaged responses to Ca injection (Fig. 3A) and ble injections of 5 mM Na alone (not shown) was seen. light (Fig. 3B) before and after GtetP-induced inhibition. 2+ We conclude that the effects of GtetP are due to GtetP The time course of inhibition was similar for Ca Page 3 of 11 (page number not for citation purposes) Depolarization (mV) Maximum Slope (mV/min) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 CONTROL INHIBITED RECOVERY 1 35 LIGHT 5 mV 250 ms 2 4 6 3dInsP 3,4 5 min GtetP Injection 2+ Gu Figure 2 anosine 5'-tetraphosphate acts subsequent to InsP -mediated Ca release during excitation. 2+ Guanosine 5'-tetraphosphate acts subsequent to InsP -mediated Ca release during excitation. Intracellular pressure injection from a microelectrode containing 25 mM GtetP decreased both the responses to a test flash and intracellu- lar pressure injection of 1 mM 3dInsP . Brackets and numbers match sets of five consecutive responses to light (1, 3, 5) or 3dInsP (2, 4, 6) averaged to produce the respective trace in the inset. 3dInsP injections were interspersed between test 3 3 flashes during the periods indicated by brackets (2, 4, 6). GtetP was injected during the period indicated by a solid bar. Aver- aged responses are shown before (1, 2), at the end of drug application (3, 4), and late in recovery (5, 6). responses and light responses, however there was some case, our results clearly show that a major component of 2+ quantitative difference: responses to light were decreased Ca -induced excitation can be blocked by a GC inhibitor. 2+ by 90% whereas responses to Ca were decreased by 60% in this experiment. In six experiments, the average inhibi- GC inhibitors act prior to the opening of cyclic nucleotide tion of the light response was 88 ± 7 % and the average gated channels 2+ inhibition of the response to Ca injection was 60 ± 27 In a final set of experiments, we tested the possibility the %. These small differences have not been analyzed fur- GC inhibitor might directly antagonize cyclic nucleotide- ther. One possibility is that the greater inhibition of the gated channels. We know of no precedent or other reason light response is indicative of a minor effect of GtetP on to suspect that GtetP would affect these channels, but it 2+ excitation upstream of InsP -mediated Ca release. In any was nevertheless important to test directly for this possi- bility. This was done by examining whether GtetP affected Page 4 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. 1 mV 200 ms 0 25 50 75 100 125 GtetP Injections B. 20 mV 200 ms 025 50 75 100 125 Time (min) 2+ Gt Figure 3 etP acts subsequent to Ca -mediated excitation. 2+ GtetP acts subsequent to Ca -mediated excitation. (A) Injection from a microelectrode containing 25 mM GtetP 2+ caused a progressive decline in the response to injection from a second microelectrode of 1.8 mM Ca solution buffered with 2+ 2 mM HEDTA. Data points are the average response with error bars (std. dev.) to ten consecutive Ca injections before, after inhibition of the light response by GtetP, and late in recovery of the light response. GtetP was injected during the period indi- cated by the bar positioned between the graphs. Brackets and arrows match response amplitude to averaged voltage time 2+ course in the insets. (B) GtetP injection inhibited the response to test flashes in parallel to the decline in response to Ca . Page 5 of 11 (page number not for citation purposes) 2+ Light Response (mV) Ca response (mV) + BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 A. B. 1.5 25 - 1.0 - * 10 - 0.5- 5 - Control GtetP Control GtetP Flash cGMP 5 mV 1 mV 200 ms 200 ms Gte Figure 4 tP acts prior to opening of cyclic nucleotide-gated channels. GtetP acts prior to opening of cyclic nucleotide-gated channels. (A) Injection from a microelectrode containing 25 mM GtetP was used to desensitize cells to a test flash by 90% (left panel). Data points are the average response with error bars (std. dev.) to seven consecutive test flashes. (B) The response to injection from a microelectrode containing 250 uM Rp- 8pCPT-cGMPS (cGMP) in the same three cells was qualitatively unaffected by GtetP (left panel). Respective responses before (control) and after injection (GtetP) are matched by lines and symbols (+, *, and open circles). The voltage traces represent averaged responses from one cell (*) to light (left) and Rp-8pCPT-cGMPS (right) before (control) and after GtetP intracellular injections. the excitation produced by intracellular injection of the for each measurement low (n < 10). In control experi- cGMP analog, Rp-8pCPT-cGMPS. We minimized intracel- ments using these conditions (not shown) the response to lular accumulation of this membrane-permeant, high- Rp-8pCPT-cGMPS, the response to light, and membrane affinity agonist by keeping the number of injections used properties remained stable over long periods. Fig. 4 shows Page 6 of 11 (page number not for citation purposes) Peak Amplitude (mV) Peak Amplitude (mV) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 2+ 2+ that the response to Rp-8pCPT-cGMPS was relatively intracellular Ca [15-17] and thwarted by Ca buffers 2+ unaffected by GtetP (10 to 30% decrease, N = 3), whereas [16,18]. Ca elevation is thus necessary and sufficient for the light response decreased enormously (90%). In two excitation. additional cells, the response to Rp-8pCPT-cGMPS injec- tion appeared qualitatively unaffected by GtetP, but prob- Several lines of work indicate that the final step is the acti- lems with clogging, a tendency of microelectrodes vation of cGMP-gated channels. Excitation can be induced containing Rp-8pCPT-cGMPS, precluded quantitative by PDE inhibitors [25,47] or by intracellular injection of analysis. cGMP [23,24]. Most importantly, cGMP can directly acti- vate channels when applied to inside-out excised mem- Discussion brane patches from the R-lobe [19]. These channels have There has been substantial previous work on the pho- properties similar to the light-activated channels in cell- totransduction cascade in Limulus, but the reactions attached patches on the R-lobe [48]. Most recently, a puta- involved in the late stages of the process have been tive cyclic nucleotide-gated channel gene has been cloned unclear. In particular, there has been no information on from Limulus [22]. The mRNA for the channel is expressed 2+ -mediated Ca enzymatic steps downstream from InsP in photoreceptors and the protein product was specifically elevation that might couple this elevation to channel localized in the R-lobe [21]. opening. Recently, it was shown that GC was required in the cascade [27], but its position in the cascade was not The work reported here shows that GC is appropriately 2+ known. Here we have demonstrated that GC is down- positioned in the cascade to couple the light-induced Ca 2+ stream from Ca and thus situated appropriately to medi- elevation to the production of cGMP. In principle, the role ate late stages of the cascade. As a result, a rather complete of GC could be simply to constitutively produce cGMP; picture of the transduction cascade is now possible. In the during light cGMP might be elevated due to a decrease in paragraphs below, we provide an overview of this cascade PDE activity. However, such a decrease in PDE activity and delineate areas where gaps remain. during light exposure would probably enhance the response to injected cGMP relative to the dark-adapted In Limulus, excitation is initiated by conversion of rho- response and certainly not decrease it, the observed effect dopsin to metarhodopsin by light (Fig. 5). The active state [24]. These results thus strongly suggest that the GC is acti- 2+ of metarhodopsin activates a G protein as evidenced by vated as a result of the light-induced elevation of Ca . the fact that G protein inhibitors decrease the light- Because there are few photoreceptors in the ventral eye, response, whereas G protein activators mimic the light this preparation is not well-suited for biochemistry to the 2+ response [31,35,36]. Metarhodopsin is inactivated in less extent that experiments to test for the Ca or light- than 150 ms [37]; while active about 10 G proteins are dependence of GC are not practical. Therefore what is turned on [38]. The G protein involved has been identi- known in these photoreceptors about GC is based on its fied as G in Limulus [39,40]. In the next stage of the cas- pharmacological profile. It has been concluded that the cade, PLC is activated by G , resulting in the hydrolysis of GC involved is not the soluble, NO-dependent form and 2+ phosphatidyl inositol-4,5-bisphosphate to produce InsP therefore does not rely on Ca -dependent activation of and diacyglycerol. PLC antagonists such as neomycin, nitric oxide synthase [27]. An important unresolved issue 2+ spermine, and U-73122 decrease the response to light is how the enzyme might be regulated by Ca . Several 2+ precedents for Ca -dependent activation of this enzyme [41,42] (see also [15]). Diacyglycerol may be important 2+ for excitation in Drosophila [10]; however in Limulus this is must be considered. For instance, in vertebrates Ca - not likely to be the case [43]. InsP has been shown to dependent GC activating proteins (CD-GCAPs) and neu- meet all the requirements for acting as an intracellular sec- rocalcin are known to activate rod GC [49,50]. The con- 2+ ond messenger necessary for excitation in Limulus: endog- centration of Ca required for this activity is well within enous synthesis, increased concentration in response to the range achieved during Limulus phototransduction light, and excitation through exogenous application [6,7]. [44,45]. In ciliates there is a form of GC that can be acti- 2+ vated by Ca /calmodulin [51]. This raises the question of 2+ InsP produces a Ca efflux from intracellular stores and whether GC activation in Limulus might be mediated by 2+ can raise cytosolic Ca upwards of 150 µ M [6,7,44,45]. calmodulin. The involvement of calmodulin in a critical Excitation by light or InsP is blocked by the InsP receptor step in the transduction cascade could be one reason for 3 3 antagonist heparin [18,29]. Direct measurements show the high concentrations of calmodulin found in Limulus 2+ that Ca release is sufficiently fast to activate the light- R-lobes [52]. dependent conductance [14,45]. The InsP receptor is localized in the endoplasmic reticulum adjacent to the The Limulus cascade is more complex than that of the ver- base of the rhodopsin-containing microvilli at the site of tebrate rod, but this increased complexity can be viewed 2+ Ca release [46]. Excitation can be mimicked by raising in light of the remarkable performance characteristics of Page 7 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 Hυ R Rhodopsin hodopsin Metarhodopsin * Metarhodopsin * G GDP GDP G GTP * GTP * PLC PLC PLC-Gq * PLC-Gq * PIP PIP InsP InsP 2+ 2+ Ca Ca ER ER 2+ 2+ Ca Ca cytoplasm cytoplasm GC GC GC* GC* GTP GTP cGMP cGMP CNGC CNGC closed closed C CNGC NGC open open A m Figure 5 odel for Limulus excitation. A model for Limulus excitation. The cascade is initiated by the isomerization of rhodopsin to metarhodopsin by light. Metarhodopsin catalyzes exchange of GTP for GDP on multiple G proteins (G ). G -GTP binds and activates phospholipase C q q 2+ (PLC). This complex cleaves phosphatidyl inositol-4,5-bisphosphate (PIP ) producing InsP . InsP opens Ca ion channels in the 2 3 3 2+ 2+ endoplasmic reticulum (ER) leading to the release of Ca into the cytosol. Ca release activates GC. A rise in cGMP opens cyclic nucleotide-gated ion channels (CNCG) in the plasma membrane. Page 8 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 Limulus photoreceptors. These cells generate single tion [15]. GtetP, HEDTA, and IBMX were obtained from photon responses in the nA range, three orders of magni- Sigma; InsP and 3dInsP from Calbiochem; Rp-8pCPT- 3 3 tude larger than those of the rod. Furthermore, Limulus cGMPS from Biolog. photoreceptors respond over nearly 4 orders of magni- tude greater range of light intensities than rods [53,54]. Microscopy The Limulus cascade has eight stages compared to the five The selection and observation of cells has been described stages of the rod cascade. The larger number of stages may in detail elsewhere [27]. Briefly, cells were observed under underlie the greater single-photon response and wider infrared illumination with Hofmann optics using a Cooke dynamic range seen in Limulus photoreceptors. Corporation Sensicam. Cells were chosen on the basis of having a stable membrane potential and robust dark Conclusions adapted and single photon light responses. Although much has been determined about the pho- totransduction cascade in Limulus, the late steps occurring In some experiments the electrodes had to be placed into 2+ between InsP -induced Ca elevation and the opening of the light-sensitive R lobe of the cells. Under Hoffman the cGMP-gated channels has been unclear. Previous work optics, the R-lobe has a smooth appearance, contrasted showed that guanylate cyclase was necessary for with the granular appearance of the light-insensitive A- generation of the light-response, but did not identify lobe. In these cases, the tip of the electrode was positioned where in the cascade it acted [27]. The major question at the border of the two lobes and advanced axially into answered in the present study is to determine whether GC the R-lobe. is appropriately positioned at the end of the cascade 2+ where it could couple Ca elevation to cGMP elevation. Intracellular pressure injection Our conclusion is that this is the case; the excitation pro- Injection electrodes were backfilled with at least 2 µL of 2+ duced by either InsP or Ca injection can be greatly solution and routinely recorded membrane potentials reduced by inhibiting GC (Figs. 2, 3). Importantly the GC approximately 20 mV higher than 3 M KCl electrodes. inhibitor did not affect the excitation produced by injec- Injections were observed on a monitor. The pulse dura- tion of cGMP analog (Fig. 4); therefore channel function tion and pressure were adjusted to maintain a constant appears unaffected. Taken together with previous results, bolus size. Injection electrodes became clogged on occa- a picture of the enzymatic steps by which rhodopsin is sion, and the blockage was cleared using either a manually coupled to channel activation in an invertebrate rhabdo- controlled high pressure pulse or brief (< 30 ms) oscilla- meric photoreceptor can now be proposed (Fig. 5). tion train. If the cell's light or drug injection responses were affected by the clearing procedure, that experiment The simplest interpretation of the available data is that GC was not used. activation is the primary means by which the intracellular concentration of cGMP is increased during excitation in Abbreviations Limulus photoreceptors. This hypothesis can be tested by A-lobe arhabdomeric lobe characterizing the specific guanylate cylase involved and 2+ the link between Ca release and cyclase activation. 3dInsP 3-deoxy-D-myo-inositol-1,4,5-trisphosphate Methods GC guanylate cyclase Electrophysiology The dissection and techniques for electrophysiology have GtetP guanosine-5'-tetraphosphate been described in detail elsewhere [16]. Cells were exposed to stimulus light with a maximal light intensity of HEDTA N-(2-hydroxyethyl)ethylenediaminetriacetic acid 1.0 mW/cm which was attenuated by neutral density fil- ND ters (attenuation = 10 ). Cells were perfused with artifi- IBMX 3-isobutyl-1-methylxanthine cial sea water (ASW) with the composition (in mM) 425 NaCl, 10 KCl, 10 CaCl , 22 MgCl , 26 MgSO , and 15 Tris, InsP D-myo-inositol-1,4,5-trisphosphate 2 2 4 3 adjusted to pH 7.8. Non-injecting intracellular microelec- trodes contained 3 M KCl (15–25 mΩ resistance). Injec- PDE phosphodiesterase tion microelectrodes contained drugs as described in the text with (in mM) 150 KCl, 10 HEPES, 0.001% Triton X- PLC phospholipase C 100 [17] and had 7–15 mΩ resistance. The microelec- 2+ 2+ trodes used to inject Ca contained 1.8 mM Ca buffered R-lobe rhabdomeric lobe with HEDTA. This use of HEDTA has been described else- where and shown to not affect excitation or light adapta- Page 9 of 11 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/7 17. 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FEMS Microbiol.Letts. 1982, 13:303-306. 52. Battelle BA, Dabdoub A, Malone MA, Andrews AW, Cacciatore C, Calman BG, Smith WC, Payne R: Immunocytochemical localiza- tion of opsin, visual arrestin, myosin III, and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors. J Comp Neurol 2001, 435:211-225. 53. Lisman JE, Brown JE: Light induced changes of sensitivity in Limulus ventral photoreceptors. J.Gen.Physiol. 1975, 66:473-488. 54. Brown JE, Coles JA: Saturation of the response to light in Limu- lus ventral photoreceptors. Journal of Physiology 1979, 296:373-392. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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