The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light

J Comp Physiol A (1992) 170:311-316 Journal of ComparaUve ~ ' ~ ' ~ m i ,

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�9 Springer-Verlag 1992

The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light Richard Payne x'* and Thomas M. Flores 2

Departments of Zoology 1 and Biochemistry 2, University of Maryland, College Park, MD 20742, USA

Accepted November 20, 1991

Summary. The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure- injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 ~ and 22 *C and with an extracellular calcium concentration of 10raM, the responses of 4 cells to light and to injections of 100 ~tM inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP3 included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature length- ened the latency of the response to light (Q10 approximately 3.2 between 7 and 22 ~ more than that to inositol trisphosphate (Q10 approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.

Key words: Phototransduction- Limulus polyphemus- Inositol trisphosphate - Calcium - Latency

Introduction

The response of a Limulus ventral photoreceptor to a single effective photon consists of a variable latent period

Abbreviations: l n s P 3 D-myo-inositol 1,4,5 trisphosphate; AS W Ar- tificial seawater; Cai Cytosolic free calcium ion concentration; Cao Extracellular calcium ion concentration

* To whom offprint requests should be sent

followed by a discrete wave of depolarization (a bump; Fuortes and Yeandle 1964). The statistical independence of a bump's waveform from its latent period and the greater sensitivity of the latent period to temperature has lead to the conclusion that the latent period and the bump waveform are controlled by separate biochemical processes (Stieve and Bruns 1983). Low extracellular calcium ion concentration (Ca0) increases the average bump latency and its variability without increasing bump duration (Martinez and Srebro 1976; Wong et al. 1982; Stieve and Bruns 1983). As a result, the latency of the response to a flash of light containing many effective photons is prolonged when Ca0 is lowered (Lisman 1976; Stieve et al. 1986). The average bump latency is also more prolonged than is the bump duration when temperature is lowered. The effects of low Cao are synergistic with those of low temperature (Martinez and Srebro 1976). Lowering both Ca0 and temperature would therefore be expected to lengthen greatly the latent period before depolarization is first detected following a light flash.

Injection of D-rnyo-inositol 1,4,5 trisphosphate (InsP3) also depolarizes Limulus ventral photoreceptors (Fein et al. 1984; Brown et al. 1984). InsP3 releases calcium ions from an intracellular store, probably smooth endoplasmic reticulum (Baumann and Walz 1989). The calcium ions released by InsP3 somehow activate a cation-selective conductance pathway in the plasma membrane which is indistinguishable from the light- sensitive conductance, thus depolarizing the photoreceptor (Payne et al. 1986a). If calcium excites the photoreceptor by acting on an intermediate in the visual cascade, then the distinction between the processes responsible for bump latency and bump waveform provides a convenient starting point for an investigation of the site of calcium's action.

Materials and methods

Experimental procedures. Conventional methods for intracellular recording and for stimulating ventral nerve photoreeeptors were

312 R. Payne and T.M. Flores: Latency of photoreceptor response to inositol trisphosphate

used, as described in detail elsewhere (Fein and Charlton 1977). Cells were stimulated with white light from a 100 W quartz-halogen source which was passed through a heat filter, neutral density (ND) filters and a solenoid-operated shutter before being focused onto the specimen plane. A 1.3 ms interval between the beginning of the electronic pulse sent to open the shutter and the opening of the shutter leaves was measured using a photodiode. The intensity of light at the specimen, with no intervening ND filters, was 80 mW/ cm 2. Light intensities are quoted in this paper as log units of attenuation relative to this intensity, which represents the delivery of approximately l0 s effective photons per receptor per second. In order to view the preparation with an infra-red-sensitive TV camera, cells were also continuously illuminated by an infra-red beam, created by passing a second beam of light from the quartz-halogen lamp through an infra-red filter before focusing it onto the specimen.

Measurement ofaequqrin luminescence. Cytosolic calcium ion concentration was monitored using the photoprotein aequorin (Shi- momura and Johnson 1962), as previously described (Payne et al. 1990). For micro-injection, the aequorin was dissolved at a concentration of 6.7 mg/ml in carrier solution containing 100 I~M EG- TA. Each photoreceptor was injected with up to 20 injections of 1-10 pl of aequorin solution, with several seconds allowed between each injection to avoid damage to the photoreceptor due to the injection of excessive volumes. Light from the preparation was collected by an objective lens (L25 x FL, 0.36 NA, Leitz, Wetzlar, F.R.G.) and projected onto a dichroic mirror (DC675LP, Omega Optical Inc. Brattelboro VT) mounted at 45* to the light beam. Infra-red light passed through the dichroic mirror and was focused onto the infra-red-sensitive TV camera. Visible light, including aequorin luminescence, which was reflected from the dichroic mirror, was focused onto the photocathode of a photomultiplier tube (R464, Hamamatsu Corp., Bridgewater N J). TTL pulses from the output of a photon-counting amplifier/discriminator (3470/AD6, Pacific Instruments, Concord CA) were counted, placed into time bins and continuously displayed by an IBM AT computer. Aequor- in luminescence is expressed in the text as photon counts per second (c.p.s.). In order to record aequorin luminescence after a stimulating light flash, an electronic pulse was delivered to open a shutter in front of the photomultiplier 10 ms after a 30 ms stimulating light flash had been delivered to the preparation. The shutter used (model 225L, Vincent Assoc., Rochester, NY) has a total opening time of 6 ms, resulting in an overall delay of 46 ms between the onset of the stimulating flash and the acquisition of any aequorin luminescence. The peak aequorin luminescence recorded after a bright flash ( I = - 1 log units, 20 ms duration) varied between 56,000 and 174,000 c.p.s. These figures are comparable to recordings published by O'Day and Keller (1989) and Bolsover and Brown (1985), who used a similar aequorin method. Records of membrane potential, pulse monitors and aequorin luminescence were simultaneously acquired, displayed and digitally stored using a personal computer running the ASYST programming language. Analysis of the response latencies and display of records was also achieved using ASYST.

The ventral nerves were pinned into a plexiglass chamber, vol- ume 0.5 ml, and ASW was passed through the chamber at a rate of 5 ml/min. Prior to entering the chamber, the ASW was cooled with a Peltier device (Interconnection Products Inc., Pompano Beach, FL). A miniature copper-constantan thermocouple was placed as close to the nerve as possible and a digital thermometer (model DP30, Omega Inc, Stamford, CT) was used to record the temperature within the ASW.

Rapid pressure injection of substances into cells through single barreled micropipettes was achieved as previously described (Cor- son and Fein 1983), with the following modification. The electronic valve that switched pressure to the micropipette was placed as close to the micropipette holder as possible (within 5 cm). A miniature pressure transducer was placed between the valve and the micropipette holder, linked to both by polyethylene tubing.

Estimation of the delay before fluid ejection from the micropipette. The time delay between the electronic pulse sent to the valve and the ejection of fluid from the pipette tip was measured by recording the change of resistance at the pipette tip when a pulse of ASW was ejected into a bath of distilled water. Resistance between two micropipette tips placed within 5 ~tm of the pressure-injection micropipette was measured using a bridge circuit. Eight ms after the beginning of the electronic pulse sent to the valve, pressure began to rise in the tubing connected to the micropipette. The resistance between the recording electrodes began to fall within 20 ms of the beginning of the electronic pulse sent to the valve, indicating the ejection of fluid. The resistance reached a minimum within 50 ms of the beginning of the electronic pulse sent to the valve.

Chemicals and solutions. InsP3 was obtained from Calbiochem (San Diego, CA) as the trilithium salt. All chemicals injected into cells were dissolved in carrier solution (100 mM potassium aspartate, 10 mM HEPES, pH 7.0). EGTA, potassium aspartate and aspartic acid were obtained from Sigma Chemical Corp (St. Louis, MO). All inorganic reagents were of analytical grade. The calcium concentration in injected solutions was measured using an ion-selective elec- trode (Ammann et al. 1987).

Cells were normally bathed in artificial seawater (ASW) containing (in mM) 435 NaCI, 10 CaC1 z, 10 KCI, 20 MgC1 z, 25 MgSO 4 and 10 HEPES, pH 7.0. 0 Ca-ASW was made by replacing the CaC12 with 1 mM EGTA obtained from Sigma Chemical Corp.

Recombinant aequorin was the generous gift of Drs. O. Shi- momura (Marine Biological Laboratory, Woods Hole, MA), Dr. S. Inouye (Chisso Chemical Corp., Yokohama, Japan) and Dr. Y. Kishi (Dept. of Chemistry, Harvard University, Cambridge MA). Recombinant aequorin was made by incubating recombinant apoaequorin (Inouye et al. 1985, 1989) with coelenterazine (Kishi et al. 1972; Musicki et al. 1986).

Results

Latency of depolarization following light flashes or brief injections of InsP 3

In all o f the exper iments o f this pape r , in ject ions o f so lu t ions con ta in ing InsP 3 were m a d e into the l ight- sensit ive, r h a b d o m e r a l (R-) lobe o f the p h o t o r e c e p t o r , the lobe o f the cell k n o w n to con ta in InsP3-sensi t ive ca lc ium stores whose release depo la r izes the cell by opening ca t ion channels in the p l a s m a m e m b r a n e (Payne and Fe in 1987). The pos i t i on o f the R- lobe was conf i rmed by scanning the cell wi th a smal l spo t o f l ight, while recording the depo l a r i za t i on p r o d u c e d by l ight flashes.

The r is ing phases o f depo l a r i za t i ons fo l lowing the in jec t ion o f 100 lxM InsP 3 by b r i e f (30 - 100 ms) pressure pulses o r fo l lowing br ie f l ight flashes were c o m p a r e d in the presence and absence o f ex t race l lu la r ca lc ium and at va r ious t empera tures . The response la tency was defined as the t ime t aken for the d e p o l a r i z a t i o n caused by ei ther l ight o r InsP 3 to exceed the r .m.s b a c k g r o u n d electr ical noise by a fac tor o f 3. The r.m.s, noise was typ ica l ly 200 ~tV. The response la tency was m e a s u r e d f rom the t ime a t which an e lec t ronic c o m m a n d pulse was sent to open e i ther the shut te r o r the pressure valve connec ted to the microp ipe t te . The la tency therefore includes a 1.3 ms de lay before the shut te r opened and a 20 ms de lay before fluid was ejected f rom the mic rop ipe t t e (see M e t h - ods). F o r the cell o f Fig. 1, the responses to InsP 3 and l ight had a s imi lar l a tency at 21 ~ and in n o r m a l A S W

R. Payne and T.M. Flores: Latency of photoreceptor response to inositol trisphosphate 313

2 0 m V

5 0 0 m s 7 0 0

600

500

,400

300

200

Latency (ms)

ASW

lOO

o

A

I i i i i i i

9 11 13 15 17 19 21

Temperature (~

Fig. 1A-H. Depolarizations of a Limulus photoreceptor in response to pulsed intracellular pressure injection of a solution containing 100 gM InsP3 or to light flashes. In each record, A-H, the response nearest the arrow is that to InsP 3. The bars below the records show the times at which a 50 ms duration light flash, intensity - 5 logt0 units, or a 100 ms duration pulse of InsP3 were delivered. Records A-C show responses obtained when the photoreceptor was bathed in ASW, at the following temperatures: A, 21 *C; B, 14 *C; C, 10 ~ Records E--H show responses obtained when the photoreceptor was bathed in 0 Ca-ASW, at the following temperatures: E, 21 *C; F, 15"C; G, 11 *C; H, 21 *C, recorded after G. Trace D shows the result of a control injection of InsP 3, delivered 10 s after the photoreceptor was desensitized by a 10 s duration exposure to bright light, intensity - 2 logto units

Table 1, Temperature-dependence of the latency of depolarization induced by light flashes or by injections of InsP3. Latencies of responses of several cells recorded within the indicated temperature ranges were pooled and averaged. All latencies are quoted as mean • S.E.M.

LIGHT InsP3 LIGHT InsP3 18-22 ~ C 18-22 ~ C 7-10 ~ C 7-10 ~ C latency (ms) latency (ms) latency (ms) latency (ms)

10Ca 71• 41 56• 270• 149• 0 Ca 108• 103 59• a 442+394 105+ 94

1 13 determinations from 4 cells 2 6 determinations from 4 cells 3 7 determinations from 3 cells * 6 determinations from 3 cells

(Figs. 1A and 2A). R e d u c i n g the t empera tu re , while m a i n t a i n i n g Ca o at 10 m M , l eng thened the la tency o f the response to l ight m o r e t h a n tha t to InsP 3 (Figs. 1 B, C and 2A). In o r d e r to de t e rmine the m a g n i t u d e o f any ar te fac- tual d e p o l a r i z a t i o n induced by the phys ica l act o f pressure inject ion, in ject ions o f InsP 3 were de l ivered 10 s a f te r l ight a d a p t a t i o n b y a p r o l o n g e d b r igh t l ight. The cell 's r esponse to InsP 3 was then inh ib i ted (Brown et al. 1984; Fe in et al. 1984) and no a r t e fac tua l d e p o l a r i z a t i o n was obse rved (Fig. ID) . In 3 o the r exper iments , the la-

O Ca-ASW

Latency (ms) 7 0 0

600 o

500 o

4OO

300

2OO O

loo |

0 i i i i i i i

7 9 11 13 15 17 19 21

B Temperature (~

Fig. 2A, B. Temperature-dependence of the latency- of responses to InsP3 and light. The latencies of responses of the photoreceptor used in Fig. 1 to injections of solution containing 100 IxM InsPa (squares) and to light flashes (circles) are plotted against temperature when the photoreceptor was bathed in ASW (A) and 0 Ca-ASW (B)

tency o f the response to InsP3 was found , on average , to be less t han tha t to l ight over the r ange 22 to 8 ~ (Tab le 1). The d a t a o f Tab le 1 p r o v i d e a Qlo for the l a tency o f the response to InsP3 o f a p p r o x i m a t e l y 2.3 over the r ange 22 - 7 ~ i f the d e a d - t i m e o f the p ressu re valve is ignored . I f the d e a d - t i m e is sub t r ac t ed f r o m the latencies , the Q~o rises to a p p r o x i m a t e l y 3. The Q~0 o f the l a tency o f the r e sponse to l ight was a p p r o x i m a t e l y 3.2.

W h e n the p h o t o r e c e p t o r o f Fig. 1 was b a t h e d in 0 C a - A S W at 21 ~ the la tency o f the r e sponse to l ight was p ro longe d , b u t l i t t le effect was no ted on the l a tency o f the response to InsP 3 (Figs. 1E a n d 2B). The p e a k d e p o l a r i z a t i o n fo l lowing in jec t ion o f InsP 3 also inc reased s l ight ly in 0 C a - A S W , as p rev ious ly n o t e d (Brown and R u b i n 1984; P a y n e et al. 1986b). L o w e r i n g o f free cy to - solic ca lc ium ion c o n c e n t r a t i o n (Cal) du r ing pe r fus ion wi th 0 C a - A S W pr io r to the in jec t ion o f InsP 3 w o u l d reduce f eedback inh ib i t i on o f InsP3- induced ca lc ium release by e levated C a i (Payne et al. 1990).

3/4 R. Payne and T.M. Flores: Latency of photoreceptor response to inositol trisphosphate

Lowering the temperature while the cells were bathed in 0 Ca-ASW markedly increased the latency of the light response compared to that of the response to InsP3 (Figs. IF, G and 2B). The timecourse of the response to light became irregular due to the dispersion of the latencies of individual bumps. Compared to the results obtained when the cell was bathed in ASW, the temperature-dependence of the light-response increased in 0 Ca-ASW, while that to InsP3 was unaffected. Results from 3 cells are summarized in Table 1. As noted by others (Martinez and Srebro 1976), the effects of lowered extracellular calcium and of lowered temperature on the latency of the light response were not additive. Lowering both temperature and extracellular calcium produced a much greater prolongation of the latency of the light response than that predicted by the sum of the prolonga- tions produced by either treatment alone. The Q10 of the latency of the response to InsP3 in 0 Ca-ASW, corrected for the dead-time of the pressure valve was approximately 2.0 in the range 2 2 - 7 ~ while that to light was approximately 3.4.

The effects of lowered extracellular calcium and of temperature were reversible. As an illustration of the reversibility of the effects of temperature, we show in Fig. 1 responses at 21 ~ recorded in 0 Ca-ASW before (Fig. 1 E) and after (Fig. 1 H) lowering the temperature to 11 ~ The responses in Fig. 1A-D, recorded in normal ASW, were made after the responses in Fig. 1E-H, de- monstrating reversal of the effects of 0 Ca-ASW on the latency of the light response.

The ability of reduced Ca0 to prolong the latent period of the light response is thought to be caused by a reduction in cytosolic calcium ion concentration. Using ion-selective intracellular electrodes or aequorin, a reduction of cytosolic calcium ion concentration has been observed in photoreceptors bathed in 0 Ca-ASW (Bol- sover and Brown 1985; Levy and Fein 1985; O'Day and Gray-Keller 1989). Modulation of the response latency

20 mV

3000 c.p.s. [

500 ms

Fig. 3A-D. Light- and InsP3-induced depolarization and aequorin luminescence recorded at 21 *C (A and B) and 11 ~ (C and D) from a photoreceptor bathed in 0 Ca-ASW. In each record (A-D), the response nearest the a r r o w is that to InsP3. Depolarization (A) and aequorin luminescence (B) simultaneously recorded at 21 ~ following a 30 ms duration injection of solution containing 100 laM InsP3 or by a 30 ms duration light flash, intensity - 3.5 logto units. De- polarization (C) and aequorin luminescence (D) simultaneously recorded at 11 *C following the same light flash or injection of InsP3 as was used in A and B. The bars below the aequorin traces indicate the times at which the light flashes or InsP3 injections were delivered

by intracellular calcium ions has been inferred from iontophoretic injection of Ca/EGTA solutions into the photoreceptors, which increases or decreases the latency depending upon the proportion of Ca mixed with the EGTA (Martinez and Srebro 1976). We therefore thought it possible that the insensitivity to Ca o of the latency of the response to InsP 3 might be due to a contamination of the injection solution with calcium. We therefore added lmM EGTA to the 100 gM InsP 3 in the solution injected into 3 cells, reducing the injected free calcium ion concentration to 2 x 10 -s M. The results obtained with the modified InsP3 solution were very similar to those of Table 1 [data not shown]. Calcium contamination therefore is unlikely to explain the lack of an effect of Cao on response latency. In order to determine whether the injected concentration of InsP3 had an effect on the latency of the response to InsP3, we also repeated the experiment of Fig. 1 for 3 cells injected with a solution containing 10 laM InsP3 instead of 100 laM InsP3. The latencies of responses to 10 p.M InsP3 and to light in these experiments were very similar to those of Table 1 [data not shown].

Latency of aequorin luminescence following light flashes or brief injections of I n s e 3

In order to determine directly the effect of temperature on the latency of calcium release by InsP3 and light, we monitored cytosolic calcium ion concentration by mea- suring light emitted by the calcium-sensitive photoprotein aequorin (Shimomura et al. 1962). We injected cells with aequorin (see Methods) and recorded luminescence following injections of InsP 3 and light flashes at two different temperatures. In order to equalize the peak aequorin luminescence observed after a light flash to that observed after an injection of InsP3, the light intensity had to be increased by approximately 1 log unit compared to the values used in the cells of Table 1, resulting in shorter latencies of the light-response at all temperatures. The cells were bathed throughout the experiment in 0 Ca-ASW to ensure that the recorded changes in aequorin luminescence resulted solely from the mobili- zation of intracellular calcium. As noted by others (Bol- sover and Brown 1985; O'Day and Gray-Keller 1989), no baseline aequorin luminescence was observable in darkness from cells bathed in 0 Ca-ASW. In darkness a photon count rate of 11 + 3.7 c.p.s was recorded from the instrumentation in the absence of a ventral nerve. The average photon count rate in darkness when the micro- scope objective was focused on the aequorin-filled cell of Fig. 3 was 104-4 c.p.s.

Lowered temperature had a much greater effect on the latent period of the aequorin luminescence that followed a light flash than it did on that following an injection of InsP3 (Fig. 3). The latencies of the luminescence or depolarization were defined as the time taken for either signal to rise to more than 3 times the r.m.s noise level before the flash was delivered. When temperature was lowered from 20 *C to 12 ~ the latency of the InsPa- induced aequorin luminescence increased from 51 4- 7 ms

R. Payne and T.M. Flores: Latency of photoreceptor response to

(mean+ S.E.M.; 3 cells) to 107 + 6 ms, while the latency of light-induced aequorin luminescence increased from 85 4- 6 ms to 2194-41 ms. Similar results were obtained using two other ceils. This result directly demonstrates that light-induced calcium release, as well as depolarization, displays a longer, more temperature-sensitive latent period than InsP3-induced calcium release.

We also investigated the relationship between the latency of the InsP3-induced or light-induced aequorin luminescence and the latency of the corresponding depolarization. As found previously (Brown and Rubin 1984; Payne et al. 1986b), we were unable to reliably detect aequorin luminescence preceding the depolarizations induced by InsPa (Fig. 3). The rise of aequorin luminescence lagged the InsP3-induced depolarization in 2 of the 3 cells investigated, but preceded it in one cell. The mean latency of the InsP3-induced depolarization was 514-7ms at 21 ~ and 87+35ms at 12~ The InsP3- induced aequorin luminescence was therefore, on average, coincident with the depolarization at 21 ~ but the InsP3-induced luminescence lagged the depolarization by 204- 35 ms at 12 ~

In the case of the response to light, the light-induced aequorin luminescence always began to rise after the depolarization (Fig. 3). The latency of the light-induced depolarization was 45 + 6 ms at 21 ~ and 128 4- 12 ms at 12 ~ while that of the light-induced aequorin luminescence was 854-6 at 21 ~ and 2194-41 ms at 12 ~ The light-induced aequorin luminescence therefore lagged the depolarization by 40 =k 7 ms at 21 ~ and 91 + 29 ms at 11 ~

Discussion

Depolarization and the release of calcium following an injection of InsP3 lack the calcium-sensitive latent period that preceeds the response to light. This result has im- plications for two problems associated with the actions of InsP3 in Limulus ventral photoreceptors. First, how does InsP3 excite the photoreceptor? Secondly, is InsP3, a viable candidate for mediating the release of calcium and the depolarization caused by light?

Present evidence favors the idea that the calcium released by InsP3, rather than InsP3 per se, activates the light-sensitive conductance and thus depolarizes the photoreceptor. In support of this hypothesis, intracellular injection of calcium chelators abolishes the response to InsP3 (Rubin and Brown 1985; Payne et al. 1986b) while injection of Ca~ rapidly depolarizes the photoreceptor (Payne et al. 1986a). The principal objection to this hypothesis is raised by our, and others', failure to detect reliably aequorin luminescence before the InsP3-induced depolarization (Fig. 3; Brown and Rubin 1984; Payne et al. 1986b). However, if the interaction between released calcium and the mechanism responsible for the depolarization were almost instantaneous, the average 20 ms lag in the aequorin luminescence at 12 ~ might be caused by the slow response of aequorin to calcium (a half rise time of at least 12 ms at 10 ~ Hastings et al. 1969) and the 20 ms integration time used by the photo-

inositol trisphosphate 315

multiplier. In addition, since we could not detect any background luminescence due to the resting level of Cai prior to InsP3 injection when the cells were bathed in 0 Ca-ASW (Fig. 3), it is possible that an initial portion of the InsP3-induced rise in Cai went undetected.

The mechanism by which calcium might activate the light-dependent ion channels is unknown. Calcium might directly gate the opening of the light-sensitive channels, but calcium-activated channels have not been observed in excised plasma membrane patches taken from the R-lobe (Bacigalupo et al. 1991). Alternatively, calcium might activate an intermediate at some earlier stage of the visual cascade. Our results suggest that this intermediate would then have to be downstream from the latency-generating mechanism, perhaps part of the mechanism that generates the bump waveform. Calcium released by InsP3 might, for instance, rapidly modulate the synthesis or degradation of a ligand that opens the light- sensitive channels (O'Day et al. 1991).

InsP3 is produced in invertebrate photoreceptors by light (Brown et al. 1984; Szuts et al. 1986; Devary et al. 1987; Brown et al. 1987). Our observation that the latency of the depolarization and calcium-release induced by InsP~ is similar to, or less than, that induced by light indicates that InsP3 is sufficiently rapid in its action to contribute possibly to the light-induced depolarization. However, our experiments do not allow us to definitively determine whether or not InsP3 is actually the cause of the light-induced depolarization. The most straightfor- ward interpretation of the substantial time delays before aequorin luminescence was detected after the light- induced depolarization began is that the initial voltage change cannot be mediated by InsP3-induced calcium release. More complex models of phototransduction have been proposed in which, in addition to producing InsP3, light also results in the production of another messenger, possibly cyclic guanosine monophosphate (Johnson et al. 1986), which directly opens the light- sensitive ion channels (Bacigalupo et al. 1991; O'Day et al. 1991). However, before reaching this conclusion one must consider the limitation of aequorin luminescence as a measure of the initial timing of the rise in Cal. This limitation is illustrated clearly in the experiments in which we measured the aequorin signal and the membrane depolarization caused by injection of InsP3. As we note above, there are sound reasons for believing that the depolarization caused by InsP3 is mediated by an elevation of Cai (Payne et al. 1986b). Yet, in many cases, the aequorin signal only started to rise after the membrane potential. Our poor detection limit therefore weakens the conclusions that can be drawn from the differences in the timing of the initial elevation of Cal and the light-induced depolarization. A more complete description of the timing of the rise in Ca~ at different light-intensities and temperatures, using a calcium indicator that reliably de- tects resting Ca~, might yield more conclusive evidence for the necessity of a messenger of excitation other than calcium ions.

The difference in the temperature-sensitivity of the latent period of the electrical response of Limulus photoreceptors to light and to injected InsP 3 is reminis-

316 R. Payne and T.M. Flores: Latency of photoreceptor response to inositol trisphosphate

cent of the electrical response of Xenopus oocytes to acetylcholine and to injected InsP3 (Miledi and Parker 1989). Thus long, temperature-sensitive latent periods may be a general feature of responses mediated by recep- tors in the plasma membrane, such as rhodopsin and the muscarinic acetylcholine receptor, that are coupled to calcium release via GTP-binding proteins.

Acknowledgements. We thank Drs. Ed Johnson, Peter O'Day and Edith Suss-Toby for helpful comments on a draft of this manuscript and Ms. Janice Briscoe for technical assistance. This work was supported by N.I.H. grant EY 07743 and the Alfred P. Sloan Foundation.

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Documents

The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light