INTERMEDIATE PROCESSES IN PHOTOTRANSDUCTION: A STUDY IN DROSOPHILA MUTANTS

Photorhumisrry und Photobiology Vol. 32. pp. 553 to 562 0 Pergamon Press Ltd 1980. Prinled in Great Britain

INTERMEDIATE PROCESSES IN PHOTOTRANSDUCTION: A STUDY IN

DROSOPHILA MUTANTS

BARUCH MINICE* and ELI ARMON Department of Physiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel, and

The Max-Planck-Institut fur Biologische Kybernetik, 74 Tubingen, W. Germany

(Received 18 April 1980; accepred 28 April 1980)

Abstract-A variety of procedures were used to modify the light response of Drosophilu photoreceptors in order to find out if these manipulations produce effects that mimic some aspects of light adaptation. The ultimate goal of our approach is to use these experimental manipulations to dissect the different stages of the phototransduction process.

The means which were used to modify the light response were as follows: (a) light adaptation of normal photoreceptors, (b) exposure of normal photoreceptors to various levels of C 0 2 (c) light adaptation of the trp Drosophila mutant in which the receptor potential decays to baseline during illumination, (d) exposure of the temperature sensitive norpAHS2 mutant of Drosophila to elevated temperatures. Intensity-response functions were obtained using intracellular and extracellular recordings. The maximal response amplitude (V ,J was then plotted as a function of the light intensity (u) which evoked a response of half maximal amplitude. This plot was used to compare the effects of the procedures described above. Qualitatively all the manipulations had similar effects, they reduced V,,, and shifted u to higher levels of light intensity, however, quantitatively the various effects were different.

The finding that each experimental manipulation gave a different V,,, versus u plot suggests that they affect different stages in phototransduction. We suggest that the slope of the V,,, versus u function can be interpreted in terms of an ordered cascade of events in phototransduction. This interpretation might give us a tool to distinguish between early and late stages in phototransduction.

INTRODUCTION

Several means are available by which one can alter the waveform of the photoreceptor light response. The commonly used way is to expose the photoreceptor to a physiological stimulus that is a , background light. The effects of a background light on the receptor potential are: (1) to decrease the amplitude of the light response by decreasing the gain, i.e. the light induced current per absorbed photon (Lisman and Brown, 1975); this reduction in sensitivity is manifested in a shift of the intensity response function ( V - log I curve, V being the amplitude of the voltage response and I the intensity of the light stimulus) to a higher level of light intensities (Norman and Werblin, 1 9 7 G i n the vertebrates; Fein and Charlton, 1977; Dorrscheidt-Kafer, 1972; Laughlin and Hardie. 1978-in the invertebrates), (2) to reduce the noise of the steady state response by reducing the size of the unitary potentials ('quantum bumps') that sum to produce the receptor potential (Dodge el d., 1968; Wu and Pak, 1978) and (3) to decrease the response latency and rise time (Fuortes and Hodgkin, 1964; Millecchia and Mauro, 1969).

Several artificial procedures are also available by which one can reversibly alter the receptor potential and thus mimic several of the above phenomena which are normally induced by a background light.

*To whom all correspondence should be addressed.

For example, the effect of an artificial increase in the intracellular free Ca2+ ions in the Limulus ventral photoreceptors on sensitivity and time scale is qualitatively similar to that of an adapting light (Lisman and Brown, 1975), Elevated concentrations of intracellular CaZt ions shift the intensity response function to higher levels of light intensity (Fein and Charl- ton, 1977). The shift of the intensity response function by background light is a property which enables the photoreceptors to respond with high sensitivity to small changes in light intensity over a large range of background lights (Norman and Werblin. 1974).

An elegant way of influencing the characteristics of the receptor potential in a reversible manner is available in the use of single-gene mutations of Drosophila. The most useful mutants are those which have a normal receptor potential under a given environmental condition which becomes abnormal when one parameter in the environment (temperature. background light, etc.) is changed. In these mutants it is therefore possible to observe the effect of the mutation on the receptor potential in a controlled and graded manner. The Drosophila mutants that we used were: (1 ) the t r p (transient receptor potential) (Cosens and Manning. 1969; Minke rt a/.. 1975b) in which the receptor potential decays close to baseline during continuous illumination (see Fig. 3). (2) the other mutant was the r ~ o r p A " ~ ~ mutant (no receptor potential) (Deland and Pak, 1973: Pak et ul.. 1976) in which the receptor potential can be reversibly abolished by elevated tem-

553

554 BARUCH MINKE and ELI ARMON

peratures. We used these mutants in order to partially induce defects in the receptor potential and we compared the effect of these defects t o the effect of background light on the response. Furthermore, we inter- compared the mutant-induced defects in the receptor potential in order to find out whether they affect the receptor potential at the same o r at different stages in the transduction process.

One can partially, or completely, block the receptor potential of the fly a t an intermediate stage in the phototransduction process by anoxia and C 0 2 (Wong et al., 1976). W e also compared the partial receptor potential block of normal Drosophila photoreceptors by C 0 2 t o the effect of the mutations and background light on the response.

The primary question we had in mind throughout this study was: do all of these manipulations block phototransduction a t the same or a t different stages?

MATERIALS AND METHODS

The experiments were performed in normal* white-eyed in viuo preparations of Drosophila. The preparation of the fly for the electroretinogram (ERG) and intracellular recordings has been described elsewhere (Wu and Pak, 1975; Minke ef a/., 1975a). In several experiments where long stable recordings under C 0 2 application or at elevated temperature were required we used only ERG recordings. However, in the normal eye, the receptor com- ponent of the ERG is considerably distorted by a contri-

*We call a fly normal 'when its photoreceptor characteristics are indistinguishable from those of wild type fly.

RELATIVE I N T E N S I T Y (log I)

-7.0

i_r--- - 5 8

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-3 8 -

I ,-

bution from the second order lamina neurons (Goldsmith and Bernard, 1974; see also Fig. 1). Therefore, we used for the ERG recordings a special mutant called ora (outer rhabdomere absent, Koenig and Merriam, 1976) in which the peripheral (R1-6) photoreceptors in each ommatidium do not have their rhabdomeres and only the central photoreceptors (R7/8), which do not synapse in the lamina, are functioning. The ERG of the ora mutant has a receptor potential which is very similar to that recorded intracellularly (Wu and Wong, 1977). Figure 1 (left) illustrates a sample of ERG recordings from w; ora mutant (white-eyed ora) in response to increasing intensities of light. The right traces are ERG recordings from normal Drosophila for a comparison. The figure shows that the ERG recorded from the ora mutant, which arises from receptors 7/8 only, has a shape typical for intracellularly recorded receptor potential (Alawi and Pak, 1971) while the ERG of the normal fly has a different shape due to contribution of lamina neurons. The figure also shows that the relative light intensity needed to elicit a criterion response is about 100 times stronger for the ERG that arises from R7/8 receptors. We used for our recordings the triple mutants w; t r p ora and w norpAHS2; ora and the double mutant w; ora.

The arrow in Fig. 1 (middle trace of the left column) indicates a secondary peak that we used to measure peak amplitude in our ERG measurements in the ora mutant. We could not use the higher initial peak transient since we observed in many cases that this phase became regenerat- ive with a spike-like potential in the ora mutant (but not in normal fly in intracellular recordings).

For stimulating light we used a Braun photographic flash in conjunction with an OG510 edge filter (Schott). One flash was strong enough to put the pigment system into photoequilibrium. We also used long pulses of XBO 150 W Xe light source with light intensity attenuated with neutral density filters, and sometimes together with a monochromatic 524 nm green interference filters (Schott- depal). The unattenuated 524 nm green light intensity was 6 x 10'' photons/m2/s.

R E L AT IV E INTENSITY

(log 1 )

U -4.9 -

1 7 Figure 1. A comparison between the ERG of normal fly to that recorded in the ora mutant. Left-ERG recordings from a dark adapted ora mutant (white-eyed fly) in response to increasing intensities of white light (as indicated). In this mutant only R7/8 are functioning. The ERG that arises from these receptors is not distorted by contributions from second order neurons and reflects the shape of the receptor potential. The arrow indicates the secondary peak transient which was used to measure response

amplitude. Right-ERG recordings from normal Drosophila when R1-6 and R7/8 are functioning.

Intermediate processes in phototransduction 555

RESULTS

Measurements of the intensity response functions in normal photoreceptors

The basic function that we measured was the intensity response function ( V - log I curve) under various experimental conditions (see Figs 2, 4 and 5). We could always fit this function with the hyperbolic function of the form:

where V is a peak response amplitude at any given stimulus intensity I, V,,, is the maximum response amplitude at a specific condition and u is the value of the intensity which evoked a half-maximum response amplitude (Naka and Rushton, 1966). The exponent n usually has a value of 0.5-0.6 for both the ERG and the intracellular recordings. Ocasionally, n has value

as low as 0.4 (see Fig. 4B) and as high as 0.75 (see Fig. 2A). In several experiments, especially when we used intracellular recordings we did not measure the responses to the highest intensities corresponding to V,,,. The reason was to avoid movement of the prep aration which might result in damage to the cell from which we had to measure the family of V- log 1 curves (see for example Fig. 2A). We found that the values of V,,, could be predicted very accurately by fitting the hyperbolic function with least squares devi- ation to the experimental points. We found that using this procedure we can estimate V,,, to within S;;. We shall show that the manipulations which resulted in modifications of the receptor potential can be separated into different categories according to their effects on the relation between u and V,,,.

Figure 2 illustrates a family of V - log I functions measured from the peak transient of the intracellularly recorded receptor potential (2A-from a peri-

r- Extracellular

/ B

-LOG RELATIVE INTENSITY

Figure 2. An example of two families of intensity-response functions which were measured intracellularly (A) and extracellularly (B). (A) All the points were measured at the peak of the intracellularly recorded receptor potential of a single R1-6 photoreceptor in normal Drosophiltr in response to increasing intensities of 524 nm monochromatic green light pulses (Schott depal interference filter). The upper curve was measured in the dark-adapted cell and the rest of the curves were measured during a 524 nm background light with log relative intensities of - 6.25, - 5.25. -4.25, respectively. The smooth curves were calculated using equation 1 with n = 0.60, 0.70, 0.75. 0.75 for the upper to the lower curves. respectively. The values of n, u. and V , , , were chosen to give best fit to the experimental points. In the curves obtained with background lights. only the increment intensities were plotted. The figure shows that background light increases u and reduces V,,,. (B) V - log I curves obtained by ERG recordings of normal receptors in an ora mutant in response to increasing intensities of white light. The upper curve was measured in a dark-adapted state while the rest of the curves were measured during white background light with relative log light intensities of - 6.15. - 5.10 and -4.15. respectively. As in Fig. 2A only the incremental responses to incremental intensities of white light were measured. The continuous curves in Fig. 2B are the hyperbolic function with n = 0.6. for the upper three curves and n = 0.7 for the bottom curve. We found in our experiments that n has values between 0.4-0.75 with no consistent dependence on the way of recording or the type of cells used. Figure 2 (A.B) shows that similar families of V - log I curves are obtained with either intracellular recording from receptors R1L6 or with ERG recordings from the ora mutant.

In all the figures the V - log I curves obtained under normal conditions in a dark adapted state (the control curves) were superimposed at the same position on the relative light intensity scale.


pheral R1-6 photoreceptor in normal fly) and from the secondary peak of the ERG (see arrow in Fig. 1, left column) from the central photoreceptors R7/8 in the ora mutant (2B). Both families of functions were derived from normal photoreceptors which were either in a dark-adapted state (upper curves in 2A, B) or in light-adapted states (other curves). The light adapted responses were measured at various intensities of background lights. The figure shows that the V- log I curves of both receptor types (R1-6, R7/8) form one family of curves with no curve cross- ing another. In both, there is a reduction in V,,, and a shift of CJ to higher light intensities with increasing light adaptation. Throughout this paper, we will refer to the V- log I curve of a normal photoreceptor without distinguishing the different receptor type or the recording technique (see Fig. 6). We plot in Fig. 2 the incremental response as a function of the incremental light intensity. The reduction in V,,, at low and medium background lights is mainly caused by response compression resulting from the steady-state response to the background light which reduces V,,,, the maximal voltage response, by decreasing the voltage range available for the incremental responses (Boynton and Whitten, 1970). The maximal voltage range available for the receptor potential of the normal fly during the background light is indicated by the short response to maximal intensity yellow flash superimposed on the background light in Fig. 3. This range is reduced (smaller response to the strong flash)

when the steady state phase is larger (see also Laugh- lin and Hardie, 1978). At higher intensities of adapting lights another mechanism may cause the reduction in V,,,. This mechanism may be a reduction in quantum efficiency for quantum bump production (Dodge et al., 1968; Wong, 1978; Wu and Pak, 1978; Minke and Kirschfeld, in preparation).

The effect of the trp and norpA*” mutations and of COz on the V - log I curve

One of the most interesting phototransduction mutants of Drosophila is the trp mutant (Cosens and Manning, 1969; Minke et al., 1975b; Minke, 1977, 1979), the intracellularly recorded receptor potential of which is shown in Fig. 3 where it is compared to the receptor potential of a normal Drosophila. For very dim lights the response seems normal but a t slightly elevated light intensities the response quickly declines to baseline (and even below baseline) during the light. The response of the normal photoreceptor (Fig. 3) is very different since it has a pronounced steady state level which continues as long as the light is on.

The bottom trace of Fig. 3 shows by a bridge measurement that in the t rp mutant the decay of the potential during light probably arises from a conductance decrease, as one would expect from an adaptation mechanism which decreases conductance to sodium ions.

trp Mutant Normal Relative Intensity Relative Intensity

I (log 1)

iomV

R

1 2 1

Figure 3. The difference between receptor potentials of the f r p mutant and of normal fly. All traces are intracellularly recorded receptor potentials for various intensities of 524 nm green light stimuli in individual R1-6 photoreceptors of a trp mutant (left) and normal white-eyed fly (right). The short duration response to a maximal intensity yellow flash (OG 510 edge filter) superimposed on the steady state response in the normal fly, shows the voltage range still available for the response. The bottom trace on the right column is a bridge measurement in the t r p mutant. The figure shows that the receptor potential of the t r p mutant in response to continuous illumination is very different from that of normal photoreceptors since it decays to.baseline during illumination while the response of the normal fly does not. The relative intensity, log1 = 0 of Fig. 3 corresponds to relative intensity. log1 = -2.65 in the

other figures.


Figure 4A shcws V - log I curves measured intracellularly in single R1-6 receptors of the frp mutant while dark adapted and during various levels of dim background lights (the rest of the curves in Fig. 4A). The effect of a dim background light in the mutant (Fig. 4A) is a pronounced decrease in V,,, with relatively small shift in 6. Here we do not attribute the decrease in V,,, to a reduction in the voltage range available for the response as in the normal fly (Fig. 3), since in the mutant brighter background lights give no steady state response. In both Figs 2 and 4A the background light depresses V,,, to a high degree. In the normal fly (Fig. 2) the background light has to be increased - 100 times more than in the mutant in order to reduce V,,, to the level obtained in Fig. 4A.

Figure 4B shows a family of V - log I curves of the normal R7/8 receptors as measured by the ERG in the ora mutant under COz. The figure compares the dark adapted curve to similar dark adapted curves measured in the same fly but with two concentrations of C 0 2 (Fig. 4B). The effect of C 0 2 is clearly to reduce V,,, and to shift o slightly. The decrease in

V,,, can, at least partially, be attributed to the induction of depolarization by the C02 (Wong et al., 1976; Lantz and Mauro, 1978) which reduces the voltage available for the response.

In a manner similar to the experiments illustrated in Fig. 4 we measured (see Fig. 5 ) V - log I curves of mutant R7/8 receptors by recording the ERG of the w n0rp .4"~~; ora mutant. In this temperature-sensitive mutant elevated temperatures (34°C) for short times (< 30 s) abolish the receptor potential reversibly (Deland and Pak, 1973). Longer exposures to high temperatures cause an irreversible change in the receptor potential, which becomes very slow (Wu and Won& 1977; Pak et al., 1976). The intracellularly recorded response from this mutant was first demon- strated by Pak et al. (1976). Figure 5 shows that these mutant flies initially show a normal V - log I curve when dark-adapted. The fly then received a prolonged heat treatment (37°C for 90 s in the middle curve and 36°C for 120s in the bottom curve) and then the V - log I curve was measured - 10 min after each heat treatment in a dark-adapted state. The heat

trp Mutant

tw[ A f r

Lu

I-

Lu w

5 4 3 2 ; - L O G RELATIVE INTENSITY

I+,,,,// O x ) 9 8 7 6 5 J 3 2

- L O G RELATIVE INTENSITY

Figure 4. Comparison of the V - log I curves measured in single R1-6 photoreceptor of a rrp mutant during various intensities of background lights (A) as compared to similar curves measured in normal dark adapted receptors under increasing concentrations of C o t (B). A-All the points were derived from intracellular recordings of the receptor potential in response to increasing intensities of 524 nm green light. The upper curve was measured in the dark-adapted cell and the rest of the curves were measured during 524 nm background light with log relative intensities of -6.65, -6.25, -5.95. respectively. The figure shows that relatively dim background light (log I = - 5.95) caused a large decrease in V,,,. (see lower curve). In the normal fly (Fig. 2A) a similar reduction in V,,, required a background intensity - 100 times more intense. The smooth curves were calculated from the hyperbolic function which best fits to the experimental points with n = 0.6 in the upper three curves and n = 0.5 in the bottom curves. B-V - log I curves measured by the ERG from normal receptors 7/8 in single ow mutant in response to increasing intensities of white light pulses. All the curves were measured in a dark-adapted fly. The upper curve was measured in normal (no CO,) conditions, and the other two curves during application of two different concentrations of CO, mixed with oxygen. The continuous lines are hyperbolic functions which best fits to the experimental points with n = 0.40. 0.45. 0.50 for the upper. middle and lower curves. respectively. This figure shows that the effect of CO,, is manifested in a reduction in Vn>.,, and a

shift in cr qualitatively similar to the effect of background light in normal photoreceptors (Fig. 2).


r-- norp A H52 Mutant

i

-LOG RELATIVE INTENSITY

Figure 5. V - log I curves measured in the narpAHSZ mutant in response to white light stimuli at 18°C. All the curves were obtained from ERG recordings from receptors 7/8 in an individual w n o r p A H S 2 ; ora mutant.

The upper curve was measured under normal conditions with no heat treatment. The rest of the curves were obtained from a dark-adapted fly at 18°C following heat treatment of 37-C for 90 s and 36°C for 120 s in the middle and lower curve, respectively. The continuous curves are the hyperbolic functions with n = 0.50, 0.60, 0.65 for the

upper, middle and lower curves, respectively.

treatment induced an irreversible change in the waveform of the receptor potential, which became un- usually slow, especially when the light was turned off (Pak et a/ . , 1976). The V - log I curves which were always measured after the heat treatment showed a reduced V,,,, which became smaller when the amount of heat given was increased. Only a small shift in a was observed (see below).

Analysis: changes of V,,, as a function ofa.

Vmax, CJ and n are the parameters which characterize a V - log I curve. We found that n did not change consistently under the various experimental conditions. Therefore, in order to compare the effects on the V - log I curves of the several procedures that we used, we plotted V,,, as a function of o for the families of V - log I curves which were obtained under the various experimental conditions illustrated in Figs 2,4 and 5.

Qualitatively it seems from Figs 4 and 5 that application of C 0 2 at various concentrations and the acti- vation of the trp and norpAH5’ mutations to various degrees by light and elevated temperatures respectively causes effects similar to those of light adaptation: both manipulations induce a clear reduction in V,,,, and a shift in o to higher intensities.

Can we conclude from the above observations that COz and trp and norpAH5’ mutations mimic some important aspects of light adaptation? Furthermore, we know that high background lights in the trp mutant, high temperatures in the norpAHSZ mutant, and high concentrations of COz in the normal fly can completely block the receptor potential in a reversible way, a t an intermediate stage of phototransduction (Minke, 1979 in the trp; Deland and Pak, 1973; Pak et al., 1976 in the norpAHsz;’ Wong et al., 1976 with COz). Can we determine whether all of these procedures block transduction at the same or at different stages?

In the trp mutant we already have evidence (Minke et al., 1975b) that the decay of the potential during background light does not arise from ‘normal’ light adaptation, namely from a decrease in the size of the quantum bumps. The decay of the potential seems rather to arise from a reduction in the quantum effi-

I! \ Y

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U/6 Figure 6. A plot of the V,,, vs u function obtained from families of I/- log I curves in the trp mutant (m) and normal photoreceptors (0). The rrp points were derived from three different flies in which the responses from six cells were intracellularly recorded. The ERG was recorded from two mutant flies. The points from the normal photoreceptors were derived from two cells recorded intracellu- lady in two flies and seven flies recorded by the ERG. The continuous lines are regression lines which best fit to the two clusters of points. The upper left star (*) icdicatesn all the figures) the control point for u and Vma.(u and V,,,, respectively). It was obtained in the dark-adapted flies. The rest of the points obtained under various intensities of background lights were normalized relative to this point. The figure shows that there is a very significant difference between the effect of background light in the normal photoreceptors and the mutant photoreceptors. Note that the first upper point of the mutant fits better the ‘normal’ regression line-a fact which suggests that under very dim

background light the trp mutant behaves normally.


E >

025-

Figure 7. A plot of the V,,, vs cr function in three oru mutants under various concentrations of C 0 2 . The continuous upper curve is a regression line which was fitted to the V, , , versus u function measured in normal photorecep tors during various background lights. The figure shows that the effect of C 0 2 is different from the effect of back-

ground light on the V- log I curves.

ciency for bump production which does not arise from bleaching of the visual pigment (Minke et al., 1975b). In order to decide whether the observed reductions in V,,, and shifts in u induced by C 0 2 the trp and n ~ r p A ” ~ ~ mutations are related to light adaptation we plot in Figs 6,7 and 8 V,,, as a function of u. This plot also helps us to decide whether the various ways of blocking the receptor potential affect one or more stages in phototransduction. In order to facilitate the comparison of different manipulations carried out in different flies we always normalized V,,, and u in each fly relative to a control value of V,,, and o (Figs 6-10) that obtained in that fly in a dark-adapted state under normal conditions (v,,, and 0 respectively). The control was always repeated at the end of the experiment except in norpAHs2 mutant where the effect of high temperature was partially irreversible.

Figure 6 compares the Vmax versus u function measured in normal photoreceptors and the same function measured in the trp photoreceptors. In both measurements intensity of background light was the variable parameter. In spite of the scatter of points which. probably arises from the use of different individual flies, of different receptors (R1-6; R7/8) and of different ways of recordings (ERG, intracellular), very different regression lines are obtained for normal photoreceptors and mutant photoreceptors.

Figure 7 shows the V,,, vs u function plotted for normal photoreceptors under various concentrations

of C02. The upper line is a replot of the regression line of Fig. 6. The figure shows that the effect of C 0 2 is different from the effect of background light, although both cause reductions in V,,, and shifts in u.

In Fig. 8 we plot V,,, versus u for the norpAH5’ mutant. The data given by the cluster of points (Fig. 8) were obtained at 18°C in a similar manner to the data in Figs 2 and 6: families of V - log I curves were measured under various levels of background lights in mdtants which were raised in 19°C. The upper regression line is a replot from normal fly (Fig. 6). The fit of the data points to the upper regression line shows that the norpAHS2 mutant is practically normal as long as the flies are raised in relatively low (19OC) temperature. The lower cluster of points were obtained from several norpAH5’ mutants at 18°C after prolonged exposures (between 1-5 min) to various elevated temperatures (between 28-36’C). The control

norp A H52 Mutant

Without .Heating

0

0.

Figure 8. A plot of the V,,,versus u function measured in the norpAHSZ mutant in similar manner to Fig. 7. The points were thus derived from four white-eyed ora; norpAH5’ mutants (w n o r p A H S Z ; ora) during various intensities of background light at 18-C (+). The continuous line is the regression line replotted from Fig. 6 (normal fly, with background light). The cluster of points on the left (A) was derived from five dark-adapted wnorpAHs2; ora mutants at 18’C after the flies were exposed for 1-5 min to various elevated temperatures (in the range between 28-35T). The heat treatment induced an irreversible change in the receptor potential. Various background lights following the heat treatment could not give results similar to those obtained in the upper curve due to the irreversible damage caused by the heat treatment. The Figure shows (+) that the norpAHSZ mutant has light adaptation characteristics very similar to those of the normal fly when the mutant is constantly kept at 1 8 T But high- temperature treatment which reduces V , , , and shifts u

slightly, does not mimic the effect of background light.


for each temperature manipulation was the dark- adapted mutant at 18‘C without heat treatment. It is clear that the effect of high temperature in the nor- pAH5’ mutant is qualitatively similar to the effect of the rrp mutation induced by background light.

DISCUSSION

The use of V,,, cs a function. The V - log I curve has been measured in many

studies of the visual process, in both vertebrates and invertebrates. In all these studies it could be fitted by the hyperbolic function (Eq. 1). (e.g. Naka and Rush- ton, 1966; Norman and Werblin, 1974; Dowling and Ripps, 1972; Dorrscheidt-Kafer, 1972; Laughlin and Hardie, 1978).

We did not find consistent changes in the exponent n. of the hyperbolic function during the various experimental manipulations, while V,,, and a did change consistently. Therefore, we used the V,,, vs o plot in order to characterize the various effects of the experimental manipulations on the V - log I curves. Figure 6 shows that the V,,, versus u function is a sensitive way of distinguishing between the effects of light adaptation and other mechanisms which affect both u and V,,, even when both effects are induced by the same parametric change in the environment, namely the ambient light intensity.

The significant difference in the effect of background light on the V,,, versus u function for the trp mutant as compared to normal fly supports the suggestion of Minke er al. (1975b) that the decay of the potential during continuous illumination in the rrp mutant arises from a mechanism different from that of the decay of the receptor potential from the transient to the steady state phase in the normal fly. It is also interesting to note that when the trp mutant was exposed to a very dim background light (the first upper point in Fig. 6). the data point fell on the upper regression curve of the normal fly. This result suggests that for dim lights the trp mutant behaves as a normal fly and that the effect of the mutation appears only at higher levels of background light for which the receptor potential decays to baseline.

The effect of high concentrations of Ca2+ ions on the V - log I function was studied by Lipton et al. (1977) in the rods of the toad (Bufo marinus). They found that relatively high concentrations of extracellular Ca’+ ions (which presumably increase the intracellular Ca2 + concentration) greatly reduced V,,, without shifting a. A similar study in the toad rods, with apparent contradictory results, was reported by Flaming and Brown (1979). They show that high concentrations of extracellular Ca2+ ions did shift a sub- stantially.

In order to examine how sensitive is the method of plotting V,,, as a function of u in discriminating among different processes we plotted in Fig. 9 the results of Lipton et al. (1977) ( 4 6 ) and Flaming and Brown (1979) (1-3) in such a plot. In both cases a

change in the concentration of Ca2+ ions was the parameter that induced the change in V,,, and u. We also plotted in the same graph the results of Lipton et al. (1977) from the same preparation in which they used background light to reduce V,,, and shift a. Figure 9 shows that there is relatively a small difference in the effect of high extracellular Caz+ between the results of Lipton et a/. and Flaming and Brown. The effect of background light in the same experiments was considerably different from the effect of Ca2+, as indicated by the significantly different regression line that fits the points derived for various levels of background light. Recently Bastian and Fain

I A m \ . t b7 1’ \

Figure 9. V,,, versus u plot of V - log I curves measured by Lipton et a/. (1977) and by Flaming and Brown (1979) in the rods of the toad, Bufo marinus. The squares were derived from- measurements of the effect of modified extracellular concentrations of Ca2 + ions. The circles were derived from experiments of Lipton er al. (1977) in the same preparation when background light was applied. The various numbers indicate specific conditions used by the above authors in the following ways: Flaming and Brown (1979)+1) V - log I curve measured in normal conditions while the V - log I curve measured in low Ca2+ concentration is the control. (2,3) V - log I curves measured in high concentration of extracellular Ca2+ ions while low (2) or normal (3) concentration of Ca2+ are the control. Lipton er al.. (1977-4) V - log I curve (pp. 756) measured in high concentration of Ca” ions while normal conditions is the control. (5.6) V - log I curves in normal conditions and low concentration of Ca2+ is the control (pp. 754. 758, respectively). (7,8,9,10).

, V - log I curves measured during background light while dark adapted curve is a control (pp. 757,758, respectively).

The figure shows that manipulation of the concentration of extracellular Ca” ions give similar results when V , , , vs u plot is used. However, a very different result is obtained

when background light is used in the same preparation.

Intermediate processes in phototransduction 56 1

(1979) studied the effect of Ca2+ ions on the receptor potential and the V - log I curve in the toad’s rods. They also used the V,,, versus u function and found results similar to those of Fig. 9.

It Seems desirable to make measurements also in the Limulus ventral eye in which V,,, versus cr is plotted for various concentrations of intracellular free Ca2+ ions. If high intracellular Caz+ ions mimic the etrect of background light in the V,,, versus u plot, the Ca2+ hypothesis of light adaptation wiii be strengthened.

DO COZ and the trp and norpAH5’ mutations affect phototransduction at the same stage?

In many invertebrates the effect of direct photoiso- merization on the receptor potential can be separated from later processes by manipulating the visual pigment concentration between rhodopsin (R) and its stable photoproduct metarhodopsin (M).

A net photoconversion of R to M induces a prolonged depolarizing after potential (PDA) which continues in the dark long after the cessation of the light (Nolte and Brown, 1972; Hillman et al., 1972; Minke er al., 1975a; Muijser et al., 1975; Tsukahara et al., 1977; Hamdorf and Razmjoo, 1977). The PDA phenomenon is equivalent in many respects to the effect of background light with a declining intensity (Brown and Cornwall, 1975; Hamdorf and Razmjoo, 1977). Wong et al. (1976) have shown in the fly, that COz and anoxia which were applied after the induction of a PDA blocked the voltage response of the photoreceptor without blocking the excitatory process underlying the PDA, as indicated by the reap- pearance of the PDA without further illumination when anoxia was removed. Similar results were obtained by Minke (1977, 1979) in the trp and norpAHSZ mutants. In these mutants the voltage response was blocked by continuous light and elevated temperature respectively. In the mutants, the visual pigment could be converted forward from R to M and backward from M to R in a normal way when the receptor potential was blocked (Minke et al., 1975b; Pak et al., 1976). Because all the manipulations that we used in this study can abolish the receptor potential but not the process underlying the PDA*. A question arises whether application of COz and induction of the trp and the norpAHs2 mutations block the voltage response at the same or at different stages?

Figures 6,7 and 8 show that by plotting V,,, as a function of u the erects of norpAHS2 and the trp mutations and of COz can be differentiated from the effects of light adaptation on the normal photoreceptor. The figures also show that the effect of background light in the trp mutant, the effect of heat treatment in the norpAHS2 mutant and the effect of COz in normal photoreceptors are different since they show different regression lines in the V,,, versus u plot.

‘Lantz et a/. (1977) showed in the barnacle photoreceptors that the duration of the PDA is strongly affected by anoxia.

0 ’ ‘ 1 8 ‘ \ , I 1 10 102 a,d lo3 lo4

Figure 10. Vmax vs (I plot which combines all the regression lines from Figs 6 8 as indicated. The figure shows different slopes for the various lines derived from the various experiments. The line derived for the effect of background light in normal photoreceptors has a relatively small slope; heat treatment in the norpAH5’ mutant gives relative large slope, slightly steeper than the effect of trp mutation, induced by background light, and significantly steeper than the effect of COz on normal photoreceptors. We suggest that the greater slopes mean later processes in the phototransduc-

tion cascade of events (see text).

In Fig. 10 we replotted the regression lines of Figs 6,7 and 8 in one coordinate system of the V,,, versus u function for a better comparison among them. Figure 10 shows that although all the above effects reduce V,,, and change u to some degree, neither COz nor the trp and norpAHS2 mutations can mimic the effect of background light. The figure furthermore shows that although qualitatively both C 0 2 and the two mutations affect V,,, much more than cr, quantitatively their effects are different. In particular the slope in the ‘COz curve’ is similar to that of ‘normal photoreceptors’ and different from those of the mutants.

The V,,, versus u function may give us a tool to distinguish among early and late steps in the cascade of events that lead to the light induced conductance change. One may consider two extreme types of curves in the V,,, versus u plot: (a) when cr does not change, i.e. when the regression line has a maximal slope, parallel to the ordinate, (b) when V,,, does not change, i.e. the graph is parallel to the abscissa with a zero slope. In the first case each V - log I curve, in the family of curves, differes from the others only by a scaling factor. This effect can arise from any block of the ionic channels which reduces the number of available channels. Such an effect is obviously an effect on the last stage-the stage of the conductance change.


In the second case, when only n is shifted, the effect can be achieved by introducing a gray filter in front of the eye. Physiologically this effect can be obtained by accessory pigment migration. In this case, the effect is on the first step in the transduction process, the step of absorption of light by the photopigment. A prob- able explanation for the correlation between early process and small slope in the V,,, versus u function may arise from the fact that there is a large excess of photopigment molecules over the number of ionic channels (Brown and Coles, 1979). Therefore effects which reduce the number of functional pigment molecules should result mainly in a shift of 0. We suggest that various experimental manipulations which affect the V,,, versus n function can be separated as to

+This is always true in voltage clamp experiments. In our experiments where only voltage was measured, an influence on the membrane resistance outside the rhabdo- meric area could also affect the slope of the V,,, vs u function.

effects on early or late processes by the influence on the slope of the curves in the Vm,x versus u plot?. An effect on an early process will result in a curve with a small slope and an effect on later processes will result in a curve with a steep slope.

According to this analysis background light affects the response at an earlier stage than C02 and the two mutations. If the effect of CO2 is specific to a stage, then C02 affects the process a t a stage earlier than the two mutations and the trp has a tendency to affect a stage somewhat earlier than the n ~ r p A * ~ ~ mutation.

Further evidence which will support or refute our suggestions must await for future experiments.

Acknowledgements-We thank Professors A. Fein, P. Hill- man and K. Kirschfeld for valuable discussions and critical reading of the manuscript. We also thank Mrs S. Krupsky and Mr R. Beit-Ash for technical assistance.

This research was supported by a grant from the Volks- wagenwerk Foundation.

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INTERMEDIATE PROCESSES IN PHOTOTRANSDUCTION: A STUDY IN DROSOPHILA MUTANTS

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