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Spontaneous activation of light-sensitive channels in Drosophila photoreceptors.

In Drosophila photoreceptors light induces phosphoinositide hydrolysis and activation of Ca(2+)-permeable plasma membrane channels, one class of which is believed to be encoded by the trp gene. We have investigated the properties of the light-sensitive channels under conditions where they are activated independently of the transduction cascade. Whole-cell voltage clamp recordings were made from photoreceptors in a preparation of dissociated Drosophila ommatidia. Within a few minutes of establishing the whole-cell configuration, there is a massive spontaneous activation of cation-permeable channels. When clamped near resting potential, this "rundown current" (RDC) accelerates over several seconds, peaks, and then relaxes to a steady-state which lasts indefinitely (many minutes). The RDC is invariably associated with a reduction in sensitivity to light by at least 100-fold. The RDC has a similar absolute magnitude, reversal potential, and voltage dependence to the light-induced current, suggesting that it is mediated by the same channels. The RDC is almost completely (> or = 98%) blocked by La3+ (10-20 microM) and is absent, or reduced and altered in the trp mutant (which lacks a La(3+)-sensitive light-dependent Ca2+ channel), suggesting that it is largely mediated by the trp-dependent channels. Power spectra of the steady-state noise in the RDC can be fitted by simple Lorentzian functions consistent with random channel openings. The variance/mean ratio of the RDC noise suggests the underlying events (channels) have conductances of approximately 1.5-4.5 pS in wild-type (WT), but 12-30 pS in trp photoreceptors. Nevertheless, the power spectra of RDC noise in WT and trp are indistinguishable, in both cases being fitted by the sum of two Lorentzians with a major time constant (effective "mean channel open time") of 1-2 ms and a minor component at higher frequencies (approximately 0.2 ms). This implies that the noise in the WT RDC may actually be dominated by non-trp-dependent channels and that the trp-dependent channels may be of even lower unit conductance.


INTRODUCTION
Substantial evidence indicates that excitation in invertebrate rhabdomeric photoreceptors is mediated at least in part by the ubiquitous phosphoinositide cascade (Brown, Rubin, Ghalayini, Tarver, Irvine, Berridge, and Anderson, 1984;Fein, Payne, Corson, Berridge, and Irvine, 1984;Devary, Heichal, Blumenfeld, Cassel, Suss, Barash, Rubinstein, Minke, and Selinger, 1987;Bloomquist, Shortridge, Schneuwly, Pedrew, Montell, Steller, Rubin, and Pak, 1988;revs;Payne, Walz, Levy, and Fein, 1988;Nagy, 1991;Minke and Selinger, 1991). However, the final pathway of excitation, linking phosphoinositide hydrolysis and Ca ~+ release to the activation of cation channels in the plasma membrane, has remained surprisingly elusive. One reason for the uncertainty is suggested by recent evidence indicating that there are at least two functional classes of light-sensitive channel (Lima, Nasi, 1991; Limulus, Deckert, Nagy, Helrich, and Stieve, 1992; Drosophila, Hardie and Minke, 1992; Pecten, Nasi, and del Pilar Gomez, 1992). This raises the possibility that they may be activated by different mechanisms. In Drosophila, at least, any divergence would appear to be subsequent to inositol 1,4,5 trisphosphate (InsP3) production, since all responsiveness to light is abolished by mutations in the norpA gene (Pak, Grossfield, and White, 1969;Minke and Selinger, 1992), which encodes a light-dependent phospholipase C (Yoshioka, Inoue, and Hotta, 1985;Bloomquist et al., 1988;Toyoshima, Matsumoto, Wang, Inoue, Yoshioka, Hotta, and Osawa, 1990). Our own recent results indicate that one class of light-sensitive channel in Drosophila has a high selectivity for Ca 2+ ions, is blocked by La 3+ and is absent in the transient receptor potential (trp) mutant (Hardie and Minke, 1992). The light-sensitive channels remaining in the trp mutant have a much reduced permeability to Ca z+, with an ionic selectivity profile more indicative of a non-specific cation channel (Hardie and Minke, 1992). The sequence of the recently cloned trp gene (Montell and Rubin, 1989;Wong, Schaefer, Roop, La Mendola, Johnson-Seaton, and Shao, 1989) shows homologies with vertebrate voltage-gated Ca 2+ channels (dihydropyridine receptors) (Phillips, Bull, and Kelly, 1992); however, it includes only one of the four putative transmembrane domains suggesting that it encodes just one subunit of a tetrameric channel. A closely homologous gene (trpl) also expressed in Drosophila photoreceptors might encode either another class of light-sensitive channel (Phillips et al., 1992), or a second subunit of the same channel. These results suggest that the trp and trpl gene products could define a new family of channel proteins responsible for phosphoinositide-mediated Ca 2+ entry (Hardie and Minke, 1992). Very few electrophysiological studies of such Ca 2+ entry channels exist (but see Ltickhoff and Clapham, 1992;Penner, 1992, 1993), and the mechanism(s) responsible for their activation represents one of the most controversial areas of cellular Ca z+ homeostasis (reviews: Irvine, 1991Irvine, , 1992Putney, 1990Putney, , 1992Berridge, 1993).
Although some properties of the light-induced current (LIC) in Drosophila have recently been characterized (Hardie, 1991a;Ranganathan, Harris, Stevens, and Zuker, 1991;Hardie and Minke, 1992), very little is known of the properties of the underlying channels. One reason is that it has not proved possible to access single light-sensitive channels with patch electrodes in Drosophila. An alternative is to use noise and kinetic analysis of whole-cell recordings of the LIC. Normally this approach would be hampered since the LIC is dominated by the noise arising from the summation of single photon responses (quantum bumps). In this study, we report that during whole-cell recording from dissociated ommatidia there is a spontaneous activation of cation channels in the plasma membrane. We provide evidence that this "rundown current" (RDC) is mediated by the light-sensitive channels themselves, uncoupled from the transduction machinery. Two functional classes of channels can be at least partially dissected via mutation (trp) or pharmacological intervention (La3+). By determining current-voltage relationships and performing noise analysis (Colqhoun and Hawkes, 1977), we provide a preliminary description of channel properties including estimates of their effective conductance, open time, and voltage dependence.

Flies
The wild-type (WT) strain was Oregon R; for most experiments we used white-eyed (w) flies lacking all retinal screening pigments. No significant difference has been detected between these and flies with normal pigmentation. The following null mutants (all on a w background) were also used: trp TM (Cosens and Manning, 1969;Montell and Rubin, 1989) lacking a light-dependent Ca z+ channel (Hardie and Minke, 1992), and two strains both lacking the rhodopsin gene ninaE (O'Tousa, Baehr, Martin, Hirsh, Pak, and Applebury, 1985;Zuker, Cowman, and Rubin, 1985): sr Df(3R) o117 e ~ and ninaE °r~ e'/TM6. Flies were raised at 25°C. Late-stage pupae (p15) staged according to Bainbridge and Bownes (1981) or newly eclosed adults were used.

Whole-Cell Recordings
Dissociated ommatidia were prepared as previously described (Hardie, 1991a, b;Hardie and Minke, 1992) using mechanical trituration without enzymatic treatment. Whole-cell recordings were made at room temperature (19-21 °C) using boro-silicate electrodes with resistances ~ 5 M~. All recordings were from photoreceptors R1--6 (Hardie, 1985). Series resistances (R0 were in the range 7-20 MII, and 65-85% compensation was routinely used (except for noise measurements--see below). The resulting R~ errors (voltage error in mV = R~ × current) were calculated and corrected for, and in any case were < 5 mV for all quantitative data presented in this study. Data were also corrected for junction potentials (see subsection Solutions below). Illumination was via a 50-W halogen lamp, filtered by Wratten ND filters (Eastman Kodak Co., Rochester, NY), and an OG 530 yellow filter, and was delivered to the preparation by a lightguide positioned 2 cm over the bath. Log -6.0 attenuation corresponds to ~5-10 effectively absorbed photons per second (determined by direct counting of quantum bumps).

Noise Analysis
For noise analysis data were recorded without any series compensation. The current signal was filtered at 2 kHz (four-pole Bessel) and a number of records, each of 2,048 points, sampled at 5 kHz. To correct for any slight change in current during this sampling period (maximally ~ 20% change during dynamic phase of the RDC and < 5% for the steady-state used for spectral analysis), a linear regression was calculated and subtracted from each record. The power spectrum of the steady-state noise was obtained by taking the ensemble average of spectra extracted by fast Fourier transform from a number (12-48) of nonoverlapping 512-point segments of the records. The power spectra were corrected for instrumentation and recording noise by subtracting the power spectrum of noise recorded in darkness before the rundown current developed. Spectra were corrected for the filtering effect of the clamp time constant (determined from the decaying phase of the capacitive transients--range 0.3-0.9 ms) and fitted with Lorentzian functions using a Marquardt curve fitting routine (P. Fit). To minimise the filtering effect of the clamp time constant, null mutants of the rhodopsin gene ninaE were often used. RDCs in ninaE were similar to WI', but since this mutant produces a much reduced number of microvilli, the capacitance (C) of the cells (and hence the clamp time constant, Rs'C) is considerably lower than in WT.
Estimates of the amplitude of the underlying single channel events were made from the ratio of variance to mean (Colquhoun and Hawkes, 1977), and single-channel conductances calculated as chord conductances after correcting for the series resistance error and assuming the reversal potential estimated for the RDC. A significant proportion (typically 25-50%) of the variance can be expected to be filtered by the clamp time constant. This was corrected for by a factor obtained by comparing the areas under theoretical power spectra (based on the Lorentzian fits to the data) before and after convolving with the RC filtering function of the measured clamp time constant (R,-C): (1) wheref = frequency.

Rundown Is Associated with Spontaneous Channel Activation
WT. As previously reported (Hardie, 1991b), unless special precautions are taken (Hardie et al., manuscript in preparation), the LIC in Drosophila photoreceptors runs down within a few minutes of establishing the whole-cell recording configuration. When the cells are clamped near resting potential (~-60 mV), the rundown is associated with the development of a conspicuous inward current (RDC). Although the RDC will always develop spontaneously in the absence of stimulation, it can also be directly induced by bright illumination. The onset of spontaneous rundown is rather variable, occurring typically within 5-7 min, but sometimes within 1-2 min of establishing the whole-cell configuration. This is to some extent related to the access (series) resistance of the patch-pipette, but also seems to vary from one preparation to another. Rundown occurs with a variety of basic intraceUular pipette solutions including ones based on CsCI, CsC1 and TEA CI, KCI, and K gluconate and is not prevented by inclusion of ATP (4 mM) in the recording pipette or Ca 2+ buffering with 5-10 mM EGTA (pCa 7-8). Conditions which prevent rundown will be discussed in a later paper addressing possible molecular mechanisms of channel activation (Hardie et al., manuscript in preparation). Spontaneous rundown currents follow a characteristic time course ( Fig. 1 A ), accelerating over a period of several seconds, reaching a peak which can be at least 1 nA in amplitude. Invariably, this phase is associated with an equally rapid decline in the sensitivity of the cell to light (e.g., Fig. 1 C). After peaking, this inward current relaxes to a steady state which lasts indefinitely (many minutes) and is associated with conspicuous high frequency noise.
trp. In the trp mutant, which lacks one class of Ca 2+ selective channel (Hardie and Minke, 1992), significant rundown currents often fail to develop at all (22/34 cells monitored for rundown), in which case rundown is simply represented by a loss of responsiveness to light. If rundown currents do develop however, they reveal RDC recorded in adult trp at -60 mV, 0.5 mM Ca 2+ Ringer (same scale); in the interval indicated by the arrow, series resistance was checked with 10-mV voltage steps. The RDC is smaller than in WT and develops more slowly, and the bandwidth of the associated noise is greater. (C) WT photoreceptor (adult) clamped at +5 mV (~ 2 mV below reversal potential of the light response in this cell). Bath Ringer 0.25 mM Ca 2+, electrode Cs/ TEA CI. 10-ms flashes of light are repeated at 3-s intervals, intensity log -2.0, increased to log -1.0 after development of RDC (arrows). Both the RDC and the light induced currents are initially inward but then reverse in sign during rundown. The LIC reverses immediately after the RDC has reached its maximum inward excursion (arrowhead); the RDC may reverse at the same time; however, this cannot be distinguished from a simple relaxation in the absolute conductance; at the latest the RDC has reversed by the time it crosses the resting baseline (dotted line). After rundown, the reversal potential has shifted by ~ 10-15 mV so that the driving force (Eu -Erev for the outwardly directed LICs (-+8-13 mV) at the end of the trace is considerably greater than at the start ( ~ -2 mV).
significant differences from WT (c.f. Fig. 1, A and B ). Firstly, the absolute magnitude of the RDC was much smaller in trp; under similar conditions (adult flies, 0.5 mM external Ca 2+, -60 mV) peak rundown currents recorded in trp averaged 258 -+ 80 pA, n = 8; c.f 650 -+ 107 pA, n = 10 for WT). Secondly, the associated high frequency noise was more conspicuous (see below). Finally the RDC in trp develops more gradually and is not indefinitely maintained but decays to near baseline after 1 or 2 min (not shown). Further differences in voltage dependence and noise characteristics are described below.

Ionic Selectivity and Voltage Dependence of the RDC
WT. The induction by bright light, strict correlation with the decline in sensitivity to light, and alteration by the trp mutation suggest that the RDC might represent spontaneous activation of at least a subpopulation of the light-sensitive channels. This possibility was tested more rigorously by comparing the reversal potential, voltage dependence, and pharmacology of the RDC and the LIC.
To compare the reversal potential (Erev) of the rundown current and the lightinduced current, cells were clamped within a few miUivohs (< 5 mV) of Erev of the LIC so that small inward or outward currents could still be recorded in response to light flashes (e.g., Fig. 1 C). Under these conditions the RDC always developed with the same polarity (inward, n = 5 cells; or outward, n = 3 cells) as the light responses. When the cells were clamped just below E .... after a few seconds the initially inward RDC reverses in sign, becoming outward ( Fig. 1 C). Although the exact point at which the RDC reverses cannot be determined in Fig. 1 C (as the RDC presumably also relaxes during the recording), it is clear that the residual light responses also reverse their polarity over the same time period. In other words, not only are Erev of the LIC and RDC very similar, but they also show a similar shift during rundown.
Direct confirmation that the current is carried by cations was shown by substituting external cations with N-methyl-m-glucamine, which does not permeate the lightsensitive channels (Hardie and Minke, 1992) after rundown had developed. Any inward rundown current is effectively abolished under these conditions (data not shown). The current-voltage (i-V) relationship of the photoreceptors was determined by applying 50-ms voltage pulses in the range +80 to -100 mV in 20-mV steps (Fig. 2). Before rundown, photoreceptors have rather high input resistances, typically of the order of 1 G~, and the membrane is approximately ohmic when measurements are made using an internal solution consisting of CsCI and TEA C1 to block K channel activity (Fig. 2 A ). However, below ~-80 mV a slowly developing inward rectification is usually observed. This is probably mediated by chloride channels since it is absent if internal chloride is substituted with gluconate. The origin of the more rapid transient inward rectification and the slight outward rectification observed at very depolarized potentials) is unclear, although both are consistently observed in WT and trp photoreceptors. Possibly it might represent residual contribution of voltagesensitive K channels, which would generate currents in excess of 10 nA in the presence of internal potassium (Hardie, 1991b; W. Hevers and R. Hardie unpublished data). After rundown there is a dramatic change in the i-V relationship (Fig. 2, B and D). The input resistance drops to 10-100 M~ and a marked inward and outward rectification develops similar to that observed for the LIC (Hardie, 1991a;see also Fig. 4). Current responses to the instantaneous voltage steps also show characteristic transient changes which are analysed in more detail in a companion paper (Hardie and Minke, 1994). The absolute magnitude of the current can reach values > 10 nA (at +-80 mV) which is similar to the maximum light-induced current (Ranganathan et al., 1991;Hardie, unpublished data). Subtracting sets of i-V traces (as in Fig. 2 D) before and after rundown provides an alternative means of estimating Erev of the RDC (+3.8 -3.7 mV [n = 9] at 2 mM Ca; values corrected for -3-mV junction potential). Although this is some 10 mV negative to Erev of the LIC before rundown, it is indistinguishable from Erev of the residual LIC after rundown (see below; c.f. Figs. 2 D and 4 C). be estimated that 98% of the RDC has been eliminated). (D) i-V relationship of the RDC from traces in B determined from the instantaneous currents (1 ms after the voltage step; m) and the steady-state (average current between 25 and 50 ms after voltage step; l--I). The currents before rundown in A were subtracted before measurement. Data not corrected for series resistance errors (maximally +10 mV at -100 mV) or junction potential errors. All traces from the same adult photoreceptor.
To address the question of which of the two suspected classes of light-sensitive channel is responsible for the RDC in WT flies, we tested the effect of La 3+ which specifically blocks the trp-dependent channels (Hochstrate, 1989;Suss-Toby, Selinger, and Minke, 1991;Hardie and Minke, 1992). If micromolar concentrations of La ~+ are applied to the bath after development of rundown, the RDC is rapidly (1-2 rain) and almost completely blocked (> 98%) suggesting that it is largely carried by the trp-dependent channels (Fig. 2 C).
trp. Similar voltage protocols applied to trp photoreceptors after rundown reveal significant differences (Fig. 3). The inward rectification is much less apparent, and although there is still a substantial outward rectification, this develops more rapidly. Single exponentials fitted to the currents at +80 mV yield time constants (B) Current-voltage plot of the rundown current from traces in A determined from the instantaneous currents (1 ms after the voltage step; U) and the steady-state (average current between 25 and 50 ms after voltage step; V-l). As in WT (c.f. Fig. 2D) there is still a marked outward rectification, but this is now characterized by significant noise levels, and develops more rapidly. Hyperpolarizing steps induce small rapid transient inward currents, but there is little inward rectification.
As in WT photoreceptors, rundown currents in trp appear to be carried by cations: the reversal potential (determined by subtracting i-V curves before and after rundown) is close to zero (-0.8 -+ 2.3 mV [n = 15] in 0.5 mM Ca 2+ Ringer) which is similar to the Erev of the light response in trp flies before rundown (Hardie and Minke, 1992). The RDC also appears essentially the same whether recorded with Cs/TEA CI, KCI, or K-gluconate electrodes.

Residual Light Responses after Rundown
As seen already in Fig. 1 C, responses to light can usually still be elicited after rundown. However, these show a number of significant differences from normal responses recorded before rundown (Fig. 4): (a) sensitivity is reduced by at least 100-fold; (b) the responses at negative holding potentials have much slower kinetics; (c) responses no longer show the typical voltage dependent kinetics of the normal response (Hardie, 1991a); (d) the characteristic inward rectification of the normal light response (Hardie, 1991a) is absent; (e) as already mentioned, the E~ev is shifted by ~ 10-15 mV to more negative values (Er~, with 2 mM external Ca~+: before rundown 13.8 -+ 1.4 mV; immediately after rundown 2.4 -_-1.2 mV [n = 7]). In trp flies, no response to light could be detected after rundown.

Noise Analysis of the RDC
If the RDC reflects spontaneous channel activity, the noise power spectra should be fitted by simple Lorentzian functions. Analysis of the noise can then be used to  rundown. Responses normalized to response at -60 mV and corrected for series resistance errors. Note that, apart from the shift in reversal potential, the inward rectification is reduced or absent after rundown. Outward rectification is still apparent in one cell, but often could not be reliably demonstrated as sensitivity continues to decline following rundown (i-V series performed starting at -60 mV).
extract information about the underlying channel open times and conductances, although bearing in mind the uncertainty of the underlying assumptions, the accuracy of the derived parameters should be treated with some caution. WT. For noise analysis, recordings were made at -60 mV, at a variety of external Ca 2+ concentrations. To improve temporal resolution, measurements were often made in ninaE mutants where the reduced membrane surface area results in a faster clamp time constant than in WT (see Materials and Methods). As for other characteristics of the RDC, the noise properties appeared the same whether recorded with pipettes containing Cs/TEA C1, KCI, or K gluconate. Power spectra were obtained by Fourier transforming noise records sampled during the steady state of the RDC (Fig. 5; see Materials and Methods). In a few cases the spectra were well fitted by a single Lorentzian function: Power spectral density = 1/[1 + (2"rrfv) 2] ( 2) where f = frequency, and time constant, ~, was in the range 1-2 ms. This would be consistent with a single class of channel with mean open time, ~. In most cases, however, the power spectra also required the linear addition of an appropriately weighted second Lorentzian component, i.e.: Power spectral density = Wl{1/[1 + (2"rrf'ri)2]} + w~{1/[1 + (2ar72"r~)2]} Where wl and w2 are weighting factors. ~1, always the dominant component, was again in the range 1-2 ms, while "r2 lay between 0.2 and 0.5 ms. This might indicate a second class of channel or a different open state of the same channel. The slower time constant (~l) at least, showed little difference for a range of external Ca 2+ concentrations between 0 and 2 mM (see Fig. 7). Significantly, and in marked contrast to the power spectrum of light-induced noise (Fig. 5 C; Wong & Knight, 1980;Barash, Suss, Stavenga, Rubinstein, and Minke, 1988), the roll-off of the rundown noise power spectra was never seen to be steeper than a single Lorentzian. This is thus consistent with the suggestion that the RDC reflects the random openings of channels uncoupled from the amplification stages of the transduction cascade. For noise generated by random channel openings, the ratio between the variance in the noise and the mean amplitude of the current should be a measure of the underlying single channel currents (Colqhoun and Hawkes, 1977). If more than one class of channel is activated then the derived value will reflect the appropriately weighted contributions of the different channel classes. This analysis assumes that channel open probability is small, in which case the variance/mean ratio should be constant for different values of the mean. In order to obtain data at different mean amplitudes, noise was sampled and analyzed at different times during the development of the rundown current. The resulting plots of variance against the mean (Fig.  6) show that the variance/mean ratio is indeed relatively constant. An exception was sometimes found during the first second of rundown in WT (or ninaE) photoreceptors when the variance could be up to two or three times larger than expected. This might suggest that initially the noise is amplified by step(s) of the phototransduction cascade or that different classes of channels (see below) are activated with different time courses. With this exception variance/mean ratios in WT (or ninaE) photoreceptors are rather low and indicate an apparent single-channel conductance of 1.5-4.5 pS with 0.5 or 2 mM external Ca 2+ (mean 2.9 -+ 1.1 pS In = 11] at 0.5 mM Ca2+). As the external Ca z+ concentration is reduced the apparent conductance became even lower (see Fig. 7), and with nominally Ca 2+ free Ringer was only 0.7 -0.3 pS (n = 3).
trp. As is readily seen with the naked eye (Figs. 1 B and 6 A ), rundown currents in trp are characterized by more prominent high frequency noise fluctuations than in WT. This is reflected in the variance/mean ratios which suggest an average singlel channel event between 12 and 30 pS (mean = 25.8 _+ 9.5 pS [n = 8] with 0.5 mM  and La 3÷ 1.98 and 0.26 ms). The power spectrum of light-induced noise from a ~rI" photoreceptor is also shown (sampled and filtered at lower frequencies). Power is concentrated at much lower frequencies, the roll-off is considerably steeper than a single Lorentzian and has been fitted by a power of Lorentzian function: power spectral density = 1/[I + (2~rJ:'r)z] ' + with n=2.1, and -r=8.3 ms). All data recorded in 0.5 mM Ca 2+ Ringer, holding potential of -60 mV. Pipette solution KC1 (K-gluconate for ninaE fly). Ca2+; Fig. 7). By contrast, power spectra from the rundown noise in trp were very similar to those recorded in W'F (Fig. 5 C; see also Table I), with no significant differences in the derived mean open times, or the tendency to require a second Lorentzian component to fit the data. As discussed below, this raises the possibility that the rundown noise in WT is dominated by the non-trp-dependent channels and that the trp-dependent channels are essentially noise-free.
Although La 3+ applied after rundown has developed in WT almost completely blocks the RDC (Fig. 2)  . The plots are approximately linear and have been fitted by regression lines the slopes (variance/ mean) of which can be taken as a measure of the amplitude of the underlying events. In the WT photoreceptor, the variance/ mean ratios measured during the first 1 or 2 s of rundown (arrows correspond to traces 2 and 3 in A ) were higher than at later times.

DISCUSSION
Our recent study of the light-induced current in WT and trp photoreceptors suggested that at least two functional classes of channels with different ionic selectivities underlie the light response in Drosophila photoreceptors. One of these appears to be encoded, at least in part, by the trp gene and is blocked by La 3+ (Hardie and Minke, 1992;Phillips et al., 1992). In principle, however, it could be postulated that there is a single channel class that undergoes dynamic modulation in ionic selectivity. Such a hypothesis, however, would need to assume that channels constructed without the trp gene product (whose primary structure suggests it is a single subunit of a tetrameric channel; Phillips et al., 1992) have a lower Ca 2+ permeability which cannot be modulated. Furthermore, it would need to be assumed that these consequences of an altered channel subunit composition are quantitatively mimicked in the WT channel by extracellular application of La ~+. The present data, which show significant differences between the characteristics of spontaneous channel activity of WI" and trp photoreceptors add further support to the suggestion that there are indeed at least two functionally distinct light-sensitive channels in Dr0- The apparent channel conductance in WT is significantly reduced when external Ca 2÷ is lowered (value at 2 mM Ca 2÷ = 3.5 pS; in 0 Ca 0.7 pS). Data presented as means _+ SD, number of cells, n indicated on top of bars. In most cases power spectra required two Lorentzians (see Figs. 6 and 7, Table I); only the slower of the two time constants ('r], Eq. 3) is plotted.
sophila. The following discussion will assume that this most direct interpretation of the data is the case.
The RDC shows a similar absolute magnitude, reversal potential, and voltage dependence to the LIC in WT. In addition it is profoundly affected by La 3+ and the trp mutation, both of which affect one class of light-sensitive channel. This leads us to conclude that the RDC is carried by the same channels responsible, at least in part, for the LIC. The power spectra of the steady-state noise in the RDC are dominated by high-frequency components which can be adequately fitted by a single Lorentzian or the weighted sum of two Lorentzians. This is in marked contrast to the LIC, where the power density is dominated by lower frequencies. Furthermore, light-induced noise spectra show a steeper roll-off that can only be fitted by a Lorentzian raised to a power (Wong and Knight, 1980;Fig. 5), a feature which is attributed to the amplification and consequent filtering properties of the transduction cascade (Wong and Knight, 1980). The noise properties of the RDC are thus consistent with random channel openings effectively uncoupled from the transduction cascade.
Even after rundown a residual light response can be elicited in WT photoreceptors, although not in trp. Presently we cannot decide whether this is carried by trp or non-trp-dependent channels. The properties of this residual light response appear quite distinct from the normal WT light response, in particular having a negatively shifted reversal potential which might suggest involvement of the non-trp-dependent channels (Hardie and Minke, 1992). However, the RDC itself (which appears to be largely dominated by trp-dependent channels; see below) also undergoes a similar shift in reversal potential, and an alternative explanation may be a redistribution of  ; up~/w~ their relative weighting. The power spectra of two trp, one WT and one of the La3+-treated WT photoreceptors were adequately fitted by a single Lorentzian function (Eq. 2). Apparent single-channel conductances estimated in trp and La3+-treated photoreceptors are ~ 10-fold greater than in WT; however, the frequency characteristics of the power spectra are indistinguishable.
ions owing to the large currents flowing during rundown. The lack of voltage dependent response kinetics is also reminiscent of the trp phenotype (Hardie and Minke, 1992); however, this may also be misleading. The voltage dependent kinetics of the normal light response are probably mediated by sequential positive and negative feedback at a late stage in transduction (Hardie, 1991a;Hardie and Minke, 1994). A prime candidate is the InsP3 receptor (Ca 2+ release channel) which in other systems shows a bell-shaped dependency on cytosolic Ca 2+ in the submicromolar range (Baumann and Walz, 1989;Bezprozvanny, Watras, and Ehrlich, 1991;Finch, Turner, and Goldin, 1991). Presumably if Ca 2+ rises outside this range, such feedback would be circumvented.

Interpretation of Noise in the RDC
It seems likely that the RDC in the trp mutant represents spontaneous activation of the non-trp-dependent light-sensitive channels remaining in the mutant. In particu-lar, the reversal potential of the RDC is indistinguishable from that of the LIC in trp, and both RDC and LIC in trp show a marked outward rectification with little inward rectification. Analysis of noise variance suggests that these non-selective cation channels have an apparent unitary conductance as large as ~ 30 pS. An estimate for the mean open time of ~ 1-2 ms (plus a minor component at higher frequencies) is obtained from the Lorentzian fits to power spectra calculated from the steady-state noise. Some variability was observed in the apparent single-channel conductance (10-30 pS); possibly this reflects the filtering effect of the clamp time constant. Although this was corrected for (see Materials and Methods), the calculated correction factor is particularly sensitive to the estimated contribution of the second, higher frequency component of the power spectra ('r2 in Eq. 3), which could not be determined with any great confidence. The RDC in WT photoreceptors is almost completely blocked by La 3+. Although this suggests that the RDC is largely mediated by the trp-dependent channels, as discussed below, there are reasons for suggesting that the non-trp-dependent channels may be gated, directly or via an intermediate, by Ca 2+. An alternative suggestion therefore would be that both channels are activated during rundown, but that the non-trp channels require the influx of Ca 2+ via the trp-dependent channels to be activated, in which case they would also be inactivated by the La 3+ blockade. In fact, the similarity of the power spectra oftrp and WT RDCs suggests that the noise in the WT RDC may actually be dominated by the contribution of identical channels to those generating the RDC in the trp mutant, whereas essentially noise-free (low conductance) channels carry the majority of the current. This would also be consistent with the observation that the apparent single conductance decreases as extracellular Ca 2+ is lowered (Fig. 7), reflecting a decreasing contribution of the higher conductance non-trp-dependent channels. Note that since the effective channel conductance estimated in WT is ~ 10% of that in trp, if this (our favored explanation) is valid, it still suggests that at least 90% of the WT RDC is carried by trp-dependent channels. Since the absolute variance in RDC noise in trp photoreceptors is typically greater than in WT (e.g., Figs. 1 and 6), this explanation also implies that a smaller number of non-trp-dependent channels are activated during rundown in WT than in the trp mutant.
Analysis of noise variance in the RDC in WT yields a much smaller apparent single-channel conductance (= 3 pS) than in trp. If, as suggested above, this noise actually represents the contribution of non-trp channels activated by Ca 2+ influx, then the trp-dependent channels would presumably be of even lower unit conductance-at most 0.7 pS, the value estimated with Ca~+-free Ringer. Single lightdependent channels previously recorded in invertebrate photoreceptors range in size from ~6 to 40 pS (reviewed in Nagy, 1991); however, channels of the size suggested here would obviously be very hard to detect in single-channel recordings. Furthermore, in Limulus the light-sensitive channels are reported, at most, to be only weakly permeable to Ca 2+ (Deckert and Stieve, 1991) and hence may belong to a distinct class from the trp-dependent channels of Drosophila. An interesting functional precedent for channels of such low conductance is provided by the cyclic GMP-gated channel responsible for the light response in vertebrate rods (reviewed in Yau and Baylor, 1989). Here it has been argued that the low conductance (in the presence of external divalent cations) is an adaptation to improve signal-to-noise ratio, by allowing a large number of channels to be active during the light response (Attwell, 1986). Possibly more directly relevant examples include a Ca 2+ and InsP4 activated Ca 2+ channel (~ 2.5 pS) believed to be responsible for phosphoinositide-mediated Ca 2+ entry in bovine endothelial cells (Ltickhoff and Clapham, 1992), and a Ca z+ conductance believed to be activated by Ca 2+ release in mast cells (calcium releaseactivated calcium conductance or CRAC), where channel-associated noise variance was undetectable in whole-cell recordings (Hoth and Penner, 1993). In principle, an alternative explanation for the apparent small conductance of the trp-dependent channels could be that the channel open times fall outside the frequency bandwidths used in our noise analysis ( < 10 Hz or > 1000 Hz). Although we cannot exclude a larger conductance channel with open time in the microsecond range, it seems unlikely that very long open times could be involved as a slow noise component would almost certainly have been detected by eye and would be difficult to reconcile with the temporal resolution of phototransduction. It might also be argued that the Ca 2+ influx during the RDC could elicit a significant electrogenic Na+/Ca 2+ exchange current which would then represent an essentially noise-free component to the RDC resulting in underestimation of the single-channel conductance. However, such a large Na+/Ca 2+ exchange current would be unrealistic, and under conditions expected to block any possible contribution of Na +/Ca 2+ exchange, namely replacing extracellular Na + with Li + and omitting Ca 2+ (to prevent reverse Na/Ca z+ exchange), RDCs (in WT) were as large, and the variance/mean ratios ( < 1 pS) similar or smaller than in controls (data not shown).

Mechanism of Channel Activation
In trp the light response decays to baseline during continuous bright illumination (Cosens and Manning, 1969;Minke, Wu, and Pak, 1975). Since the primary defect in the trp mutant is the absence of a plasma membrane light-dependent Ca 2+ conductance (Hardie and Minke, 1992), we have previously suggested that the decay in trp represents the depletion of intracellular InsPs-sensitive Ca 2+ stores and that these normally require Ca 2+ influx via the trp-dependent channels to be refilled Hardie and Minke, 1992). This interpretation implies that the non-trp channels may be activated (possibly directly gated) by Ca 2+. This would be consistent with the suggestion that the noise in the WT RDC reflects the contribution of the non--trp channels activated by Ca 2+ influx and explain why the noise variance is reduced when extracellular Ca e+ is reduced to nominally zero levels (when we expect that the RDC is almost entirely dominated by the trp-dependent channels). On this view, the non-trp channels might represent Ca2+-activated nonselective cation channels (CAN channels; reviewed in Partridge and Swandulla, 1988).
By contrast the trp-dependent channels appear to remain highly activated in the absence of both internal and external Ca e+ and in fact appear to be subject to Ca2+-mediated inactivation (Hardie and Minke, 1994). The mechanism of their activation remains speculative and a detailed discussion, beyond the scope of the present study. We recall however, that the trp-dependent channels are highly permeable to Ca 2+ (Hardie and Minke, 1992); since they are believed to be activated by the phosphoinositide cascade, we recently suggested that their mechanism of activation may be similar to that responsible for the phenomenon of phosphoinositide-mediated Ca z+ entry in a variety of vertebrate cells (Hardie and Minke, 1992). Like invertebrate phototransduction, the mechanism underlying this process is unresolved. However, current models which attempt to explain this process, including the "capacitative Ca 2+ entry" model (reviewed in Putney, 1990Putney, , 1992, and the suggestion that Ca 2+ entry is triggered by a protein-protein interaction between the InsP3 receptor and the plasma membrane Ca 2+ channel (reviewed in Irvine, 1991Irvine, , 1992, raise possible mechanisms of excitation in invertebrate photoreceptors which had not previously been explicitly discussed. Finally, we note that an important experimental approach for future progress in the analysis of invertebrate phototransduction is likely to involve a search for agents that directly activate the light-dependent channels. The partial characterization of channel properties provided in this and the companion study (Hardie and Minke, 1993) should provide a valuable database for evaluation of the effects of agents or procedures which lead to activation of the different classes of light-dependent channels.