Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo

Abstract

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. We have recently shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLCβ activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. We show here that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCβ catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCβ catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCβ via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, we show that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCβ and IP3R found here may represent a common feature of the inositol-lipid signaling.

Keywords: Ca2+ release; Drosophila; IP3 receptor; phospholipase C; photoreceptors; phototransduction.

Figure 1.

IP₃R-RNAi retinae show virtually normal eye morphology but reduced IP₃R expression levels. A–C, SEM comparing eye of IP₃R-RNAi (B, C) and WT flies (A). Scale bars: A, B, 100 μm; C, 20 μm. Arrowheads point to corneal hairs. D, Western blot analysis of isolated retinae (20–30 retinae for each lane) of IP₃R-RNAi fly using α-IP₃R. The expression levels of IP₃R in IP₃R-RNAi retinae are compared with its expression levels in isolated retinae of WT, P[UAS:IP₃R-RNAi] (UAS), and gl3 mutant (lacking the photoreceptor cells). Relative protein amounts were quantified from band intensities. The density of each lane was divided by the density of α-Moesin (middle row) and calculated as a percentage of WT flies for each experimental run. Bottom, A histogram presenting the average of three independent experiments (mean ± SEM, n = 3); no significant difference was found between WT and UAS, (t test, p = 0.140), but a significant difference was found between WT and gl3 (t test, p = 0.037) and between WT and IP₃R-RNAi (t test, p = 0.0029).

Figure 2.

IP₃R-RNAi flies show reduced ERG response to light, which was further reduced at low external Ca²⁺, in vivo. A, Intensity–response (V-logI) curve of peak ERG response amplitudes. The different curves were measured from WT and IP₃R-RNAi flies in response to 5 s lights (mean ± SEM, t test, *p < 0.05, ***p < 0.001; n = 10). B, Representative ERG traces in response to a light pulse applied to IP₃R-RNAi and WT flies. ERG traces used in A at -log1 light intensity. The open box is the light monitor. C, Reduced ERG amplitude in response to light flash at low extracellular Ca²⁺. Top, Responses to brief saturated flashes (filled arrows) before and after pressure injection of EGTA-containing solution (arrowheads). Bottom, The same experimental paradigm was repeated in the IP₃R-RNAi fly. D, A histogram summarizing the experiments presented in C. A comparison of the response to light flash between WT and IP₃R-RNAi (mean ± SEM, t test, p = 0.470 and p = 0.0016 before and after EGTA injection, respectively; n = 10). E, Superimposed ERG traces from a WT fly in response to prolonged light pulse (used in A and B) before (black) and after (red) EGTA injection.

Figure 3.

Reduced light-induced intracellular Ca²⁺ elevation in intact photoreceptors of IP₃R-RNAi flies as measured by GCaMP6f fluorescence. A, Measurements of GCaMP6f fluorescence revealing the kinetics of light-induced increase in cytosolic [Ca²⁺]. The ordinate plots ΔF/F₀ (see Materials and Methods) as a function of time in GCaMP6f flies on WT background at 1.5 mm external [Ca²⁺] (green) and at 0 (0.5 mm [EGTA]) external [Ca²⁺] (blue) and in GCaMP6f flies on IP₃R-RNAi background at 0 (0.5 mm [EGTA]) external [Ca²⁺] (red). The plotted curves are means ± SEM. To quantify the difference between the blue and red curves, we compared the difference in fluorescence intensity at 60 and 300 ms of the blue and red curves and found that IP₃R-RNAi flies expressing GCaMP6f at 0 extracellular Ca²⁺ (red) revealed a significant smaller increase of Ca²⁺ elevation in IP₃R-RNAi flies relative to WT flies (t test, p = 0.0098, p = 0.0084 at 60 and 300 ms after light onset, respectively; n = 10). Because of the high affinity of GCaMP6f to Ca²⁺ (K_d of ∼170 nm) and the large Ca²⁺ influx at 1.5 mm [Ca²⁺]_out, the Ca²⁺ indicator reached saturation at the time of maximal fluorescence and only the initial rise of the curves should be considered. B, A time series of whole ommatidia images of control (WT background at 0 external Ca²⁺) and IP₃R-RNAi ommatidia, showing the fluorescence of the GCaMP6f during light stimulation. Scale bar, 10 μm. C, No difference was found between Ca²⁺ released from internal stores of control and IP₃R-RNAi after store depletion by Tg. Measurements of GCaMP6f fluorescence at 0 external Ca²⁺ (0.5 mm [EGTA]) following 15 min of prior incubation in standard extracellular solution (1.5 mm [Ca²⁺]) including 10 μΜ Tg in WT (blue) and in IP₃R-RNAi backgrounds (red). Incubation of >20 min with Tg of ommatidia from GCaMP6f on WT background flies abolished the increase in fluorescence (green). Tg was also included in the 0 external Ca²⁺ solution during the fluorescent measurement (n = 10). D, A time series of ommatidia images of control and IP₃R-RNAi at the conditions of C, showing the fluorescence of the GCaMP6f during light stimulation. Scale bar, 10 μm.

Figure 4.

A–C, Patch-clamp whole-cell recordings showing reduced responses to light of IP₃R-RNAi photoreceptors when Ca²⁺ is buffered by EGTA in the pipette solution. A, Representative traces from whole-cell patch-clamp recordings with and without 1 mm [EGTA] added to the pipette solution. Open boxes represent the duration of light pulses. B, Intensity–response relationship of WT and IP₃R-RNAi with and without 1 mm EGTA added into the pipette solution as indicated. Inset, The initial graph of B at higher magnification. C, A histogram comparing light responses to a constant light intensity of WT and IP₃R-RNAi photoreceptors with standard intracellular solution and when the solution contained ∼300 nm free Ca²⁺ (mean ± SEM, t test, p = 0.045; n = 5). D, E, Functional Ca²⁺ pump at the internal Ca²⁺ stores is required for normal light response while Ca²⁺ pump inhibition by Tg mimicked the phenotype of IP₃R-RNAi phenotype. D, Representative whole-cell recordings of current traces obtained from WT (top) and IP₃R-RNAi photoreceptors (bottom), before and after prior incubation (10 min) of the cells in standard bath solution (1.5 mm [Ca²⁺]) containing 10 μm [Tg]. Solid and open bars represent the time of Tg application and the light monitor, respectively. E, Histograms comparing the peak amplitude of the light-induced current of WT and IP₃R-RNAi photoreceptors before and after 10 min of incubation with Tg (mean ± SEM, t test, p = 0.0015; n = 5).

Figure 5.

The frequency but not amplitude of single-photon responses was reduced significantly in IP₃R-RNAi flies compared with WT flies when the pipette solution was buffered with EGTA. A, Representative traces of unitary current responses to single photons (quantum bumps) of WT and IP₃R-RNAi ommatidia recorded with standard pipette solution (top) or with 1 mm [EGTA] added (bottom). B, A histogram comparing the quantum-bump frequency of WT and IP₃R-RNAi photoreceptors with or without EGTA in the recording pipette (mean ± SEM, t test, p = 0.0058; n > 300 bumps for each column, n = 4). C, Distributions of quantum-bump amplitudes. The Gaussians that fit the histograms of quantum-bump amplitude distribution in E (n > 300 for each histogram, with and without EGTA in the pipette solution). D, A histogram comparing the mean quantum-bump amplitudes of WT and IP₃R-RNAi with or without EGTA in the recording pipette (mean ± SEM, t test, p = 0.00038, p = 0.00078, for WT and IP₃R-RNAi flies, respectively) as derived from bump-amplitude distributions of E. E, Histograms of bump-amplitude distribution with the fitted Gaussians presented in C.

Figure 6.

Ca²⁺ is a limiting factor, which determines the maximal ERG amplitude when reduced IP₃R levels were combined with reduced catalytic activity of PLCβ. A, Intensity–response relationship of peak ERG responses to increasing intensities of light stimulations of WT (redrawn from Fig. 2), norpA^H43, and norpA^H43;IP₃R-RNAi flies. B, Traces of ERG recordings of norpA^H43 and norpA^H43;IP₃R-RNAi in response to maximal intensity light pulses (open bar) before and after application of anoxia (see D below), which is known to robustly increase cellular Ca²⁺. C, A histogram comparing the peak amplitude of the ERG light responses of norpA^H43 and norpA^H43;IP₃R-RNAi before and after anoxia (mean ± SEM, t test, p = 0.0014 and p = 0.00035 for norpA^H43 and norpA^H43;IP₃R-RNAi, respectively; n = 10). D, Traces showing the entire experiments from which the ERG traces of B were taken. Traces of prolonged ERG recordings of norpA^H43 (top trace) and norpA^H43;IP₃R-RNAi (bottom trace) in response to maximal intensity light pulses (arrows) followed by N₂ application. Anoxia was obtained by blowing N₂ on the intact fly as indicated by the horizontal line. The additional light pulses (3rd and 4th), tested the effects of cellular Ca²⁺ elevation by the anoxia on the ERG. The amplitudes of the ERG responses to the first and the third light pulses in each trace should be compared. The light pulses applied during anoxia (2 unmarked arrows) induced only very small light responses because most TRP and TRPL channels were already open by the anoxia.

Figure 7.

Synergistic effect on the response to light between reduced IP₃R level and reduced catalytic activity of PLCβ as revealed by whole-cell recordings. A, Representative traces showing whole-cell patch-clamp recordings from norpA^H43 (black) and norpA^H43;IP₃R-RNAi (red) with and without 1 mm [EGTA] added into the standard pipette solution. B, Intensity–response relationship of norpA^H43 (redrawn from Fig. 4) and norpA^H43;IP₃R-RNAi with and without EGTA added into the pipette as indicated. C, D, Dark-bump production was virtually abolished in IP₃R-RNAi photoreceptors (C, top and middle left traces). Representative traces showing dark bumps of WT fly recorded for 1 min at 1.5 mm external [Ca²⁺] while Mg²⁺ was omitted from the bath solution. The dark bumps were recorded without (top) or with 1 mm [EGTA] (middle) in the pipette solution. The paradigm of the left traces was repeated in IP₃R-RNAi fly (C, top and middle right traces). D, A histogram presenting dark-bump frequency during 1 min recordings in WT and IP₃R-RNAi photoreceptors at the conditions of C (mean ± SEM, t test, p = 0.00598, n = 5; C, bottom line and E). Quantum-bump production of IP₃R-RNAi photoreceptors was virtually abolished when extracellular Ca²⁺ was replaced by Sr²⁺ and Ca²⁺ was buffered in the intracellular pipette solution (C, bottom left). Traces showing quantum bumps of WT in response to dim light of 1.5 effective photons/s (open box) recorded for 1 min when extracellular Ca²⁺ was replaced by 1.5 mm [Sr²⁺]_out, Mg²⁺ was omitted from the bath solution, and 1 mm [EGTA] was included in the pipette solution. Bottom right, The paradigm of the bottom left trace was repeated in IP₃R-RNAi fly. E, A histogram presenting quantum-bump frequency during 1 min recordings in WT and IP₃R-RNAi photoreceptors when extracellular Ca²⁺ was replaced by Sr²⁺ (mean ± SEM, t test, p = 0.00013; n = 5).

Figure 8.

A model explaining the mechanism of spontaneous bump generation and the effect of reduced IP₃R level and Ca²⁺ release on spontaneous bump generation (see Discussion).

Figure 9.

EM pictures and a diagram showing structural features of the signaling compartment and the localization of the signaling molecules. Top, TEM showing the microvilli composing dark-adapted WT rhabdomere. A, TEM of a cross section of a rhabdomere, which is composed of tightly packed microvilli. The elongated membrane vesicles virtually touching the base of the microvilli (arrows) are extensions of the smooth ER called SMC and constitute the IP₃-sensitive Ca²⁺ stores. B, Higher magnification of the rhabdomere at the base of the microvilli, showing the SMC (arrows). C, A diagram showing the molecular components of the signal transduction cascade of Drosophila:,1, Upon absorption of a photon (hν), rhodopsin (R) is converted into metarhodopsin (M). 2, The R-to-M photoconversion leads to the activation of heterotrimeric G-protein(G_qα,β,γ) by promoting the GDP-to-GTP exchange. 3, The GDP-to-GTP exchange in turn leads to activation of PLCβ, which hydrolyzes PIP₂ into the soluble IP₃ and the membrane-bound DAG. 4, Subsequently, IP₃ molecules (blue dots) diffuse along the microvillus and bind to the IP₃ receptor (IP₃R) located at the SMC. 5, Binding of IP₃ to the IP₃R causes release of Ca²⁺ (red dots) from the SMC and its diffusion back into the microvillus followed by binding of Ca²⁺ to both PLCβ and the TRP channel. 6, This binding either facilitates the catalytic activity of PLCβ (?) or reduces the threshold of TRP channel activation (?, see Fig. 8). 7, Two classes of light-sensitive channels, the TRP and TRPL, open by a still unknown mechanism (?) following PLCβ activation. The TRP and TRPL channel openings lead to elevation of cellular Ca²⁺. Elevation of DAG and Ca²⁺ promote eye-specific PKC activity, which regulates channel activity. PLCβ, PKC, and the TRP ion channel form a supramolecular complex with the scaffolding protein INAD, which is bound to the F-actin cytoskeleton via the NINAC protein. Bottom, Magnification of the box marked by dotted lines in the top diagram.