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Previous Article | Next Article
Volume 16, Number 9, Issue of May 1, 1996 pp. 2924-2933
Copyright ©1996 Society for NeuroscienceINDO-1 Measurements of Absolute Resting and Light-Induced Ca2+ Concentration in Drosophila Photoreceptors
Roger C. Hardie Cambridge University, Department of Anatomy, Cambridge CB2 3DY, United Kingdom
ABSTRACT
Absolute Ca2+ levels in dissociated Drosophila photoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca2+ influx that saturated the dye (>10 µM Ca2+), but lagged the electrical response by 2.8 msec. Resting Ca2+ levels in the dark, measured during the latent period before the response, averaged 160 nM in normal Ringer's (1.5 mM Ca2+). Ca2+ increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca2+ during the latent period; and (2) by using ninaE mutants with greatly reduced rhodopsin levels. Ca2+ rose linearly as a function of the time integral of the light-sensitive current with a slope of 2.7 nM/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nM/pC, indicating a 2.5-fold reduction in the fractional Ca2+ current. From these data, it can also be estimated that >99% of the Ca2+ influx is effectively buffered by the cell. In Ca2+-free Ringer's, resting cytosolic Ca2+ was reduced (to 30-70 nM), but contrary to previous reports, significant light-induced increases (~250 nM) could be elicited. This rise was reduced to <20 nM when extracellular Na+ was replaced with N-methyl-D-glucamine, suggesting that it could be attributed to Na+ influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nM.
Key words: calcium entry; inositol phosphates; phototransduction; calcium fluorimetry; INDO-1; Drosophila; photoreceptor; vision
INTRODUCTION
Phototransduction in invertebrate photoreceptors is believed to be mediated by the phosphoinositide (PI) signaling cascade (for review, see Payne et al., 1988; Minke and Selinger, 1991
subunit also almost completely abolishes the response to light (Scott et al., 1995
The ability to genetically manipulate the PI cascade has been complemented by a number of experimental assays, including the ability to measure the light-activated current under whole-cell voltage clamp using a preparation of dissociated ommatidia (Hardie, 1991
MATERIALS AND METHODS
Animals. The wild-type strain was white-eyed (w) Oregon Drosophila melanogaster; in addition, the following mutants were used (on a w background): trp301, a null mutant of the trp gene that encodes a putative light-sensitive channel subunit (Montell and Rubin, 1989Solutions. The electrode solution contained (in mM): 140 Kgluconate, 4 MgATP, 0.4 Na2GTP, 2 MgCl2, and 100 µM of either INDO-1, Mag-INDO-1, or Fluo-3. (tetrasodium salts; Molecular Probes, Eugene, OR). The bath contained (in mM): 120 NaCl, 5 KCl, 4 MgSO4, 10 N-Tris-(hydroxymethyl)-methyl-2-amino-ethanesulphonic acid, 25 proline, 5 alanine, and either 1.5 mM CaCl2 (normal Ringer's) or no added Ca2+ and 1 or 2 mM EGTA (Ca2+-free Ringer's). For some experiments (see Fig. 10), all permeant ions (Na, K, Ca, and Mg) were replaced with 150 mM N-methyl-D-glucamine (NMDG) chloride. All solutions were buffered at pH 7.15; experiments were performed at room temperature (20° ± 1°C).
Fig. 10. Light-induced Ca2+ signals and simultaneously recorded whole-cell currents (lower traces) measured in the absence of both extracellular Ca2+ and Na+. In contrast to Figure 9, the Ca2+ rise was virtually abolished when Na+ was substituted with N-methyl-D-glucamine (NMDG) with 0 Ca2+ and 2 mM EGTA applied by rapid perfusion from a puffer pipette. a, Response to a 1 sec saturating illumination in a cell perfused with NMDG solution, originally bathed in Ca2+-free, Na+-containing bath. b, Response from a photoreceptor perfused with NMDG, but this time after being initially bathed in normal Ringer's (1.5 mM Ca2+); again there was little or no increase in Ca2+. Both cells, clamped at70 mV, produced small outward currents, as NMDG does not permeate the light-sensitive channels; c, Light-induced responses from the same cell as in Figure 11b to a weak 20 msec LED flash before (left), after (right), and during (middle) rapid perfusion with the Ca2+-free NMDG solution. The cell was clamped at
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Electrophysiology and Ca2+ measurement. Whole-cell recordings from photoreceptors were made from dissociated Drosophila ommatidia, as described previously (Hardie, 1991
For ratiometric Ca2+ measurements, the dual emission dyes INDO-1 and Mag-INDO-1 were chosen because they allow high sampling rates without the requirement for mechanical chopping of the excitation beam (as with Fura-2). It was recently reported that Ca2+ measurements using INDO-1 in vertebrate photoreceptors were contaminated by a change in the binding properties of the dye induced by the measuring light (Gray-Keller and Detwiler, 1994
Background correction. Because of the relatively high UV autofluorescence of the photoreceptors (typically ~30% of total signal), accurate background correction was critical for these measurements. When using ora or ninaE photoreceptors, the background fluorescence was determined routinely for every cell after giga-seal formation, but before establishing the whole-cell configuration. However, this procedure was considered impractical for WT and trp photoreceptors, because the measuring light would have resulted in severe light adaptation before the experiment started. Consequently, background was determined separately on at least two ommatidia at the beginning and end of each preparation. The measuring light induced a small change in the autofluorescence of the photoreceptors over the first 200 msec of illumination, probably because of photoconversion of visual pigment and/or changes in mitochondrial pigments. This change (an ~6% reduction in fluorescence measured at 480 nm) would have manifested itself as a slight (~20-40 nM) increase in Ca2+ and was corrected for in measurements of light-induced Ca2+ changes in Ca2+-free solutions by subtracting an averaged background template (appropriately scaled) from the raw fluorescence traces before determining the fluorescence ratio. As additional controls, background fluorescence was measured from ommatidia after forming a seal with a dye-filled electrode and also from cells during whole-cell voltage clamp using otherwise identical electrode solutions, but without indicator dyes. Background fluorescence values under these conditions differed by <5% from values determined from intact ommatidia in the absence of electrodes. Background values varied little, apart from a slow reduction over the lifetime of the lamp: from the SD of the background fluorescence (~4% of mean), it was estimated that an error of less than ± 20 nM (SD) was introduced by this indirect method of background correction.
Calibration. Free [Cai] was calculated from the ratio (R) of fluorescence at 405 and 480 nm, using the equation:
(Grynkiewicz et al., 1985
![[Ca]=K<SUP>d</SUP>×(R−R<SUB>min</SUB>)/(R<SUB>max</SUB>−R)](/content/vol16/issue9/fulltext/2924/img001.gif)
(1) Values for Mag-INDO-1 were obtained in an analogous manner except that it was not necessary to obtain an Rmin value, as resting cytosolic Ca2+ is negligible for this low-affinity dye so that Rmin could be taken from the resting value in any given cell, and Kd was determined using a solution based on the low-affinity Ca2+ buffer nitrilotriacetic acid (NTA): 10 mM NTA: 3 mM Ca (yielding 50 µM free Ca using published Kd values). When using Mag-INDO-1, Mg2+ was omitted from all solutions (bath and intracellular).
Intensities are expressed in effectively absorbed photons, calibrated, as described previously, with respect to WT photoreceptors by counting quantum bump rates in response to the dimmest stimuli and measuring the relative intensities of all other stimuli (Hardie, 1995
RESULTS
Controls
A number of control experiments were performed to establish that the indicator dye did not affect the light response, that the resting cytosolic Ca2+ concentration was stable over the time course of the experiments, and that the measuring flash did not necessarily permanently damage the cells. Ca2+ indicator dyes are themselves Ca2+ buffers and may, in principle, interfere with the kinetics of transduction, which are known to be very sensitive to cytosolic Ca2+ levels (Hardie, 1991
Fig. 1. Indicator dyes do not significantly influence the light response. a, Quantum bumps recorded in response to a 1.5 sec stimulus of identical intensity in a cell loaded with 100 µM INDO-1 are similar to those recorded from another cell with control intracellular solutions containing no Ca2+ buffers. b, Similarly, Fluo-3 (100 µM) has little or no effect on responses to 20 msec flash or 500 msec step of light (the difference in absolute amplitudes is well within the experimental variability). The dotted trace shows the response to a second 500 msec stimulus recorded 2 min after making a Ca2+ measurement using a submaximal 100 msec flash containing ~107 effective photons. All responses recorded in standard Ringer's solution (1.5 mM Ca2+) at a holding potential of1 (calibrated from bump counts as in a).
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Figure 2 shows records of fluorescence measured from a photoreceptor of an ora mutant that completely lacked rhodopsin. Fluorescence was measured continuously, starting shortly after making the giga-seal. Immediately after establishing the whole-cell configuration (break-in), the fluorescence started to increase, indicating loading of the cell with dye. The time course of loading was usually well approximated by a single exponential with a time constant in the range of 30-80 sec. The Ca2+ concentration, determined from the ratio of fluorescence at 405 and 480 nm after subtraction of the background immediately before break-in, remained effectively constant during this period, showing only an improvement in the signal-to-noise ratio as the total dye concentration increases. However, immediately after break-in there was an indication that Ca2+ may actually decline over a period of ~10 sec; in addition, in some cells there was a slow rise in Ca2+, typically starting after 3 or 4 min of whole-cell recording. To minimize variation attributable to the increases sometimes observed after longer periods of whole-cell recording, measurements in WT flies were routinely made between 2 and 3 min after break-in.
Fig. 2. Time course of dye loading in an ora photoreceptor. Raw traces (dotted) show the INDO-1 fluorescence measured at 405 and 480 nm starting shortly after making a giga-seal. After establishing the whole-cell configuration (at time 0), fluorescence rises with a single exponential time course (smooth curves, single exponential fits to the traces with time constant of 68 sec). After subtraction of the background fluorescence, the ratio R between fluorescence at 405 and 480 nm was used to calculate cytosolic [Ca2+] according to Equation 1. Cytosolic [Ca2+] remained stable throughout the 200 sec recording, except for an apparent reduction during the first few seconds of recording. Recording made in normal (1.5 mM Ca2+) Ringer's at
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Resting Ca2+ levels in the dark
Figure 3 shows a simultaneous recording of membrane current and INDO-1 fluorescence using 100 µM INDO-1 in a WT photoreceptor, sampled at 500 Hz. After a short latency (~10 msec), fluorescence measured at both wavelengths changed rapidly and in opposite directions (Fig. 3a) indicating a rise in Ca2+. The time course of the calculated Ca2+ signal is shown below, along with the simultaneously recorded whole-cell current. The INDO-1 signal was rapidly saturated by the large Ca2+ increase (indicated by the increase in noise) so that, arguably, the most useful information to be gained from such records is the resting level determined during the latent period (Fig. 3b). When considering data only from ``healthy'' cells, using the criteria of negligible leak currents (<20 pA at a holding potential of
Fig. 3. Light-induced Ca2+ rises measured with INDO-1 in normal (1.5 mM Ca2+) Ringer's. a, Raw traces (raw) of fluorescence (counts per second) measured at 405 and 480 nm, sampled at 500 Hz; [Ca2+], after background subtraction, the time course of the rise in Ca2+ is calculated from the ratio of the raw traces according to Equation 1 (see Materials and Methods). The simultaneously recorded light-induced current (LIC, lower trace, holding potential
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Identical measurements performed in the absence of extracellular Ca2+ (see further below) revealed a very significant drop in resting cytosolic Ca2+. In this case, however, values in ora mutants appeared somewhat lower than in WT (WT 70 ± 22 nM; n = 11; ora 28 nM ± 16; n = 36; measured at
Latency
A number of studies have addressed the question of whether the Ca2+ signal measured in invertebrate photoreceptors precedes the electrophysiological response. For example, in Limulus, the light-induced Ca2+ signal, which is mainly attributable to release, was found to lag the electrical response by ~3-20 msec, thus calling into question the hypothesis that Ca2+ release is causal for excitation (Ukhanov et al., 1995
Fig. 4. Latency of the Ca2+ response determined using Fluo-3 in normal Ringer's solution. The time course of the Ca2+ signal measured using the single-wavelength dye Fluo-3 was similar to that measured with INDO-1 (compare Fig. 3); however, a better signal-to-noise ratio can be obtained. The traces show the average of LIC (upper trace) and Ca2+ signal from eight photoreceptors in response to a 200 msec measuring flash (480 nm) containing ~108 effective photons. The current trace has been inverted for easier comparison of time courses. The first detectable rise in Ca2+ always lagged the LIC by ~3 msec (inset on expanded scale). Holding potential was
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Quantification of light-induced Ca influx
Ca2+ rises elicited by weak illumination To determine absolute Ca2+ levels reached in response to weak-to-moderate stimulation, a two-flash strategy was used similar to that recently used in Limulus ventral photoreceptors by Ukhanov et al. (1995)
Fig. 5. Determination of Ca2+ levels in response to weak illumination in normal Ringer's solution. a, A WT photoreceptor was first illuminated for 500 msec with a dim LED stimulus (~2000 photons/sec) generating an inward current of ~500 pA amplitude (dotted trace). The Ca2+ level reached during this period (530 nM) was then determined during the latent period of the response to a saturating UV measuring stimulus (50 msec, 3 × 107 effective photons). The Ca2+ signal (solid trace) is replotted on an expanded time base below. b, Ca2+ levels obtained from 19 cells (filled squares), as in Figure 5a, plotted against the total charge flowing during the 500 msec adapting step. Open square, ``Dark'' Ca2+ concentration determined identically, but without preillumination with the LED (mean ± SD of 12 cells). The data have been fitted by a regression line of slope 2.7 nM/pC with an intercept (dark resting Ca2+ level) of 161 nM. Triangles represent data determined using measurements of light-induced Ca2+ rises in ora or ninaE flies with small amounts of residual rhodopsin (Fig. 6).
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A second strategy for investigating the Ca2+ rises associated with small responses is to use mutants with greatly reduced rhodopsin levels, which consequently generate only small responses to the bright measuring light. As reported previously (Johnson and Pak, 1986
Fig. 6. Ca2+ influx measured in real time in ora photoreceptors containing residual levels of rhodopsin. UV measuring flashes of 1 sec duration delivered to ora (or ninaE) photoreceptors sometimes elicited small responses (lower traces) attributable to residual levels of rhodopsin, thus allowing direct measurement of Ca2+ influx (upper traces) during weak effective illumination. Traces from two different cells are shown using different intensities, generating responses of ~20-40 pA (left) and 200 pA (right). Quantum bump noise can be clearly resolved in these small responses: as reported previously (Johnson and Pak, 1986
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Maximum rises The Ca2+ influx occurring during the response to the measuring stimulus clearly saturates high-affinity Ca2+ indicator dyes such as INDO-1 and Fluo-3 (see also Peretz et al., 1994a
Fig. 7. Light-induced Ca2+ rise in a WT photoreceptor loaded with the low-affinity indicator dye Mag-INDO-1. In response to a saturating UV stimulus, Ca2+ rose rapidly beyond 50 µM. Data were recorded at a holding potential of
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Light-induced rise in trp
In the trp mutant, measurements of reversal potential have indicated that the Ca2+ permeability of the light-sensitive channels is severely reduced, leading to the proposal that the trp gene encodes a channel subunit responsible for the high Ca2+ selectivity of the light-sensitive channels (Hardie and Minke, 1992
Fig. 8. Ca2+ influx is reduced in the trp mutant. Measurements of Ca2+ levels in the trp mutant were made using the two-flash paradigm (Fig. 5) and plotted against the total charge carried in response to the adapting flash. In the dark (open triangle), resting Ca2+ levels in trp were indistinguishable from WT; however, in response to the 500 msec LED-adapting flash, the Ca2+ rise was significantly less than in WT (dotted line replotted from Fig. 5). The regression line through the trp data had a slope of 1.09 nM/pC (i.e., ~2.5× less than in WT).
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Light-induced Ca2+ increase in the absence of extracellular Ca2+
Previous studies reported either that there was no detectable light-induced Ca2+ rise in the absence of extracellular Ca2+ (Ranganathan et al., 1994
Fig. 9. Light-induced Ca2+ signals measured in the absence of extracellular Ca2+. Substantial Ca2+ increases were detected in every cell in response to saturating UV-measuring stimuli: Ca2+ signals (upper traces), simultaneously recorded whole-cell currents at 0 and
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Fig. 11. Summary of data collected in Ca2+-free solutions. Solid bars, Dark levels determined during the initial 20 msec of the response; open bars, levels reached after 500 msec. In the presence of external Na+, light-induced Ca2+ increases of >200 nM at both
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Initially these results appear to indicate the light-induced release of Ca2+ from intracellular stores; however, an alternative explanation may also be suggested. Drosophila photoreceptors have a powerful Na/Ca exchange mechanism (Hardie, 1995
The dark resting level of Ca2+ was also significantly increased by the NMDG substitution (from 70 ± 22 nM to 158 ± 43 nM; Fig. 11): this suggests that the Na/Ca exchange is indeed important in maintaining low Cai in Ca2+-free solutions; however, it also raises the possibility that the block of the light-induced Ca2+ rise might have been attributable to inhibition by the raised resting Ca2+. This possibility can probably be excluded because in the control (0 Ca, 120 mM NaCl) solution, depolarization caused a similar rise in the resting Ca2+ level (probably because of the voltage dependence of the Na/Ca exchange equilibrium), yet did not inhibit the subsequent light-induced increase (Figs. 9, 11).
Finally, the possibility was considered that the inability to detect significant release from stores was because of the putative light-sensitive stores becoming depleted during prolonged exposure to Ca2+-free solutions (Hardie and Minke, 1992
In summary, light-induced Ca2+ rises can indeed be detected in the absence of extracellular Ca2+; however, the most likely explanation for this rise appears to be a shift in the Na/Ca exchange equilibrium. In the absence of external Na+, the maximal rise detected is <20 nM over a time scale of 500 msec to 1 sec.
DISCUSSION
This study provides data on absolute levels of cytosolic Ca2+ in Drosophila photoreceptors and describes procedures for quantification of both dark- and light-induced Ca2+ signals in this important genetic model of phototransduction and PI signaling. The results confirm that the majority of the light-induced Ca2+ signal is attributable to Ca2+ influx; however, contrary to previous reports, significant rises can also be detected in Ca2+-free conditions, probably as a result of Na+ influx influencing the Na/Ca exchange equilibrium. With certain assumptions, the results also allow a number of useful quantitative estimates to be made, including (1) the effect of the trp mutation on the fractional Ca2+ current through the light-sensitive channels; (2) the effective buffering capacity of the photoreceptors; and (3) an upper limit on the amount of Ca2+ that might be released from internal storesa result that has some implications for the still unresolved mechanism of excitation.
Subcellular compartments
The supposed Ca2+ stores (submicrovillar cisternae, or SMC) in Drosophila abut directly against the base of the microvilli, defining a putative ``transduction compartment'' consisting of the microvilli and the subrhabdomeric space, which is an ~10-20 nm gap between the SMC and the base of the microvilli (Walz, 1982Resting levels
By calibrating in situ, using solutions with ionic strength similar to those used for the actual measurements, many of the uncertainties associated with calibration of Ca2+ indicators should have been avoided in the present study. There is, however, still some uncertainty as to whether the measured values are representative of those occurring in vivo. For example, there was an indication over the first few seconds after break-in that [Ca] actually fell from a higher level (Fig. 2). One explanation may be that the electrode solution contained virtually no Na+, whereas the photoreceptors can be expected to contain a few mM Na+, which would result in a different equilibrium of the Na/Ca exchanger. An alternative possibility is that equilibration of the contents of the cell with the electrode solution may change the effective Kd of the dye by, for example, a change in the ``viscosity factor.'' The dark, resting cytosolic Ca2+ level was found to be very sensitive to extracellular Ca2+, falling from 150 to 70 nM (30 nM in ora). Although not systematically explored, it seems likely that Na/Ca exchange may be one of the major mechanisms responsible, because in the absence of extracellular Ca2+, cytosolic Ca2+ rose to ~150 nM when extracellular Na+ was substituted for NMDG (Fig. 11). A similar effect was elicited by depolarization, which is also predicted to shift the Na/Ca exchange equilibrium because of its voltage dependence.Light-induced influx and buffering capacity
Measurements with Mag-INDO-1 indicated that Ca2+ may rise globally to levels as high as 50 µM; however, some caution should be exercised in accepting this figure. Recently, the effective single-channel conductance of the light-sensitive channels was found to increase ~10-fold in the absence of external Mg2+ (Hardie and Mojet, 1995For smaller currents, it was possible to quantify the Ca2+ rise more reliably (Fig. 5b). Comparison of this rise (2.7 nM/pC) with the predicted amount of Ca2+ influx via the light-sensitive channels provides an estimate of the effective buffering capacity of the photoreceptors, by which is understood here all mechanisms controlling cytosolic Ca2+, including Ca-binding proteins, sequestration, extrusion by the Na/Ca exchanger or other transporters, and exclusion from the bulk of the cell by diffusion barriers. The relative permeability of the light-sensitive channels for Ca (PCa:PNa) has been estimated at ~40:1 on the assumptions of the Goldman-Hodgkin-Katz (GHK) theory (Hardie and Minke, 1992
This calculation ignores the possibility that a component of the measured LIC is attributable to an electrogenic inward Na/Ca exchange current evoked by the Ca2+ influx. Although some contribution cannot be excluded, it is likely to be minor for the small currents used to determine the relationship between Cai and the influx current (Fig. 5b), because direct measurements of the Na/Ca exchange current using caged Ca2+ (Hardie, 1995
Effect of the trp mutation on Ca2+ influx
Measurements of reversal potential indicated that the light-sensitive channels in the trp mutant have a reduced Ca2+ permeability (Hardie and Minke, 1992The question of light-induced Ca2+ release: implications for transduction
Contrary to previous reports (Peretz et al., 1994aIt is of some interest to inquire how much Ca2+ would be required to generate an increase of 20 nM, as this is probably an upper estimate of how much Ca2+ is released by a saturating light stimulus. In turn, this could provide clues whether such release might be sufficient to account for excitation
By comparison, one can ask how many InsP3 receptors might be required for excitation. As in most invertebrate photoreceptors, each effectively absorbed photon in Drosophila evokes a discrete quantum bump (Wu and Pak, 1975
It should be emphasized that the above arguments assume that Ca2+ influx and Ca2+ release are buffered by the cell and detected by the indicator dye with similar efficiency. The possibility must also be considered that significant amounts of Ca2+ are released, but into a subcellular region distinct from that accessed by the light-sensitive channels and more remote from the indicator dye (assuming this to be at least partially excluded from the ``transduction compartment''). This possibility cannot be excluded, but would contravene the widely accepted view that the putative release channels and influx channels both have access to the same compartment, i.e., the subrhabdomeric space between the SMC and the base of the microvilli.
FOOTNOTES
Received Sept. 27, 1995; revised Feb. 12, 1996; accepted Feb. 15, 1996.
This research was supported by grants from the Wellcome Trust, Biotechnology and Biological Sciences Research Council, and the Royal Society.
Correspondence should be addressed to Dr. R. C. Hardie, Cambridge University, Department of Anatomy, Cambridge CB2 3DY, UK.
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