Phospholipase C-mediated suppression of dark noise enables single-photon detection in Drosophila photoreceptors

Abstract

Drosophila photoreceptor cells use the ubiquitous G-protein-mediated phospholipase C (PLC) cascade to achieve ultimate single-photon sensitivity. This is manifested in the single-photon responses (quantum bumps). In photoreceptor cells, dark activation of G(q)α molecules occurs spontaneously and produces unitary dark events (dark bumps). A high rate of spontaneous G(q)α activation and dark bump production potentially hampers single-photon detection. We found that in wild-type flies the in vivo rate of spontaneous G(q)α activation is very high. Nevertheless, this high rate is not manifested in a substantially high rate of dark bumps. Therefore, it is unclear how phototransduction suppresses dark bump production arising from spontaneous G(q)α activation, while still maintaining high-fidelity representation of single photons. In this study we show that reduced PLC catalytic activity selectively suppressed production of dark bumps but not light-induced bumps. Manipulations of PLC activity using PLC mutant flies and Ca(2+) modulations revealed that a critical level of PLC activity is required to induce bump production. The required minimal level of PLC activity selectively suppressed random production of single G(q)α-activated dark bumps despite a high rate of spontaneous G(q)α activation. This minimal PLC activity level is reliably obtained by photon-induced synchronized activation of several neighboring G(q)α molecules activating several PLC molecules, but not by random activation of single G(q)α molecules. We thus demonstrate how a G-protein-mediated transduction system, with PLC as its target, selectively suppresses its intrinsic noise while preserving reliable signaling.

Figure 1.

Dark bumps are the result of spontaneous GDP-GTP exchange on the G_qα subunit. A, Histogram plotting the mean bump rate of dark bumps of WT and G_qα¹ heterozygote (G_qα¹/+) and homozygote mutant raised either in the dark or in the light (n = 5, mean ± SEM). B, Scatter plot of dark bump rate as a function of G_qα protein levels in the membrane of different mutants and rearing conditions as in A. Note the highly significant linear relationship between these two parameters [mean ± SEM; data of G_qα protein levels in the membrane were taken from the work of Frechter et al. (2007)]. C, Whole-cell recordings of dark bumps from WT and Gβe¹ heterozygote (Gβe¹/+) mutant flies using extracellular solution with 1.5 mm Ca²⁺ and a pipette solution with either KGlu (no GDP) or KGlu (8 mm GDP) (Table 2). D, Histogram plotting the mean dark bump rate of the mutant flies at the designated conditions. Note that adding GDP to the recording pipette decreases dark bump rate (n = 5, mean ± SEM, *p < 0.05, ***p < 0.001).

Figure 2.

Reduced external Ca²⁺ eliminates dark bump activity while the amplitudes of light-induced bumps are enhanced. A, Whole-cell recordings from WT flies in total darkness and in dim light stimulation of 1 effective photon/s (EP/s) under 1.5 mm Ca²⁺ (top trace) and low Ca²⁺ conditions (bottom trace). Note that no dark bump activity is seen under low Ca²⁺ conditions, while a response to light stimulation is observed. In extracellular solution containing 1.5 mm Ca²⁺, dark bumps appear and light response is maintained. B, Whole-cell recordings from WT and Gβe¹/+ mutant flies at total darkness under 1.5 mm Ca²⁺ and low Ca²⁺ conditions. Note the summation of dark bumps (arrow). Also note the high dark bump rate of Gβe¹/+ mutant flies and the highly reduced dark bump activity under low Ca²⁺ conditions. C, Histogram plotting the mean dark bump rate of the mutant flies at the designated conditions (n = 5, mean ± SEM, **p < 0.01, ***p < 0.001). D, RMS of current fluctuation of dark and light (1 EP/s) responses, at the designated conditions (n = 5, mean ± SEM). Note, the low RMS under low Ca²⁺ compared with 1.5 mm Ca²⁺ conditions in the dark and the high RMS at low Ca²⁺ compared with 1.5 mm Ca²⁺ conditions in the light. Also, note the similarity between the RMS of Gβe¹/+ dark and WT light under 1.5 mm Ca²⁺. E, Whole-cell recordings in total darkness from Gβe¹/+ mutant flies under low Ca²⁺ conditions with intracellular solution without ATP (top trace) or with 1 mm ATP in the pipette (bottom trace) (n = 3). Note that dark bumps reappear at these conditions.

Figure 3.

Under low external Ca²⁺ conditions, elevating Ca²⁺ concentration in the patch-pipette solution results in reappearance of dark bumps in a dose-dependent manner. A, Whole-cell recordings from WT photoreceptors at total darkness and under low external Ca²⁺ conditions under various concentrations of Ca²⁺ in the recording pipette. B, Histogram plotting the mean bump rate of dark bumps at the different conditions. Note that the bump rate increased in a dose-dependent manner (n = 5, mean ± SEM).

Figure 4.

Dark bump activity is dramatically increased when omitting ATP from the recording pipette or by increasing intracellular Ca²⁺. A, Dark bump activity from WT and G_qα¹ mutant flies using 1.5 mm Ca²⁺ containing extracellular solution and pipette solution with no ATP. Note that in WT flies both the rate and amplitude are increased through time, while in G_qα¹ mutant flies dark bumps are scarce. B, Histogram plotting the mean bump rate with 4 mm ATP and without ATP in the recording pipette of WT and G_qα¹ mutant flies (the color code applies also to C and D, n = 4, mean ± SEM, **p < 0.01; ns, not significant, p > 0.05). C, Histogram plotting the mean instantaneous dark bump rate with 4 mm ATP and without ATP of 10 s intervals (n = 4, mean ± SEM). Note the increase in the mean instantaneous dark bump rate in WT flies when ATP is omitted from the recording pipette solution while, no change in the instantaneous dark bump rate when 4 mm ATP is added to the recording pipette solution. D, Dark bump rate is increased when adding 1 mm Ca²⁺ to the recording pipette solution under 1.5 mm external Ca²⁺ conditions compared with normal pipette solution. E, Histogram plotting the mean bump rate with 1 mm and without Ca²⁺ in the recording pipette (n = 4, mean ± SEM, **p < 0.01; ns, not significant, p > 0.05). F, Whole-cell recordings from WT flies under low Ca²⁺ condition and the effect of perfusion with Ca²⁺ containing extracellular solution. Quantum bumps produced by light stimulation of 10 EP/s at both conditions is also demonstrated. Note that no dark activity is seen under low Ca²⁺ conditions while a response to light stimulation is observed. In extracellular solution containing 1.5 mm Ca²⁺, dark bumps appear at a higher than normal rate and a light response can hardly be detected. Also note that addition of 1.5 mm Ca²⁺ induced a small outward current due to temporary reverse activation of the electrogenic Na⁺-Ca²⁺ exchanger, which transports Ca²⁺ into the cell.

Figure 5.

The G_qα¹mutant fly shows highly reduced sensitivity to light at low Ca²⁺ compared with 1.5 mm Ca²⁺ conditions. A, Intensity–response (R–logI) relationship of WT (triangles) and G_qα¹ mutant (circles) flies under low Ca²⁺, 1.5 mm Ca²⁺ and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution. Note that the WT (R–logI) relationship was shifted to the left when measured under low Ca²⁺ conditions compared with 1.5 mm Ca²⁺ while, R–logI relationship of the G_qα¹ mutant was shifted to the right. Addition of 1 mm Ca²⁺ to the recording pipette solution partially rescued the low sensitivity of the G_qα¹ under low Ca²⁺ conditions. Moreover, note that at extremely high intensities of 1.5 × 10⁷ EP/s under low Ca²⁺ condition the response amplitude of G_qα¹ photoreceptors is very small. Inset: Intensity–response (logR–logI) relationship (n = 5, mean ± SEM). B, A representative light-induced response of WT and G_qα¹ mutant to 10³ and 10⁶ EP/s respectively, under 1.5 mm Ca²⁺, low Ca²⁺ and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution. C, Western blot analysis of heads homogenate of dark raised WT, G_qα¹, G_qα¹/+ and norpA mutant alleles (norpA^P24, norpA^P57, norpA^H43) using specific Drosophila antibodies for G_qα and MOESIN as indicated. Bottom panel shows a histogram plotting the relative density of the corresponding G_qα bands of the different fly strains (n = 3, mean ± SEM).

Figure 6.

The norpA^P57 hypomorph show highly reduced light sensitivity under low Ca²⁺ conditions compared with 1.5 mm Ca²⁺. A, A model of the initial stages of phototransduction underlying dark bump production (top) and quantum bump production (bottom). B, Western blot analysis of heads homogenate of dark raised WT, norpA mutant alleles (norpA^P24, norpA^P57, norpA^H43) and G_qα¹ mutant using specific Drosophila antibodies for NORPA and MOESIN as indicated. Bottom panel shows a histogram plotting the relative density of the corresponding NORPA bands of the different fly strains (n = 3, mean ± SEM). C, Dark bumps are observed in norpA^P57 at total darkness under 1.5 mm Ca²⁺ conditions (top trace). Low Ca²⁺ condition abolished the dark activity (middle trace). Addition of 1 mm Ca²⁺ to the recording pipette solution resulted in the reappearance of dark bumps under low Ca²⁺ conditions (bottom trace) (n = 4). D, Intensity–response (R–logI) relationship of norpA^P57 mutant flies under low Ca²⁺, 1.5 mm Ca²⁺ and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution. Note that under low Ca²⁺ conditions drastically reduced the light response, compared with 1.5 mm Ca²⁺ while addition of 1 mm Ca²⁺ to the recording pipette solution rescued the low sensitivity under low Ca²⁺ conditions. Moreover, note that at extremely high intensities of 1.5 × 10⁷ EP/s under low Ca²⁺ conditions the response amplitude of norpA^P57 photoreceptors are very small. Inset: Intensity–response (logR–logI) relationship (n = 5, mean ± SEM). E, A representative light-induced responses of norpA^P57 mutant to 10⁴ EP/s under 1.5 mm Ca²⁺, low Ca²⁺ and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution.

Figure 7.

The norpA^H43 mutant with low PLC catalytic activity shows no dark bumps, reduced quantum bump rate and amplitude, and a highly reduced sensitivity to light under low Ca²⁺ conditions. A, Dark bumps are not observed in norpA^H43 at total darkness under 1.5 mm Ca²⁺ conditions (top trace) and under 1.5 mm Ca²⁺ conditions with the addition of 1 mm Ca²⁺ to the patch-pipette solution (bottom trace) (n = 4). B, Light-induced bumps in norpA^H43 mutant flies in response to light intensity of 10 EP/s (top trace) and in WT flies to light intensities of 10 EP/s (middle trace) and 1 EP/s (bottom trace) (n = 4). Note that the light-induced bumps of the mutant are small, while their kinetics are as fast as those of WT bumps. C, Histogram plotting distribution of bump amplitude of WT dark bumps, WT quantum bumps and norpA^H43 quantum bumps (n = 117). Inset, Gaussian fits to the amplitude distribution histograms (mean peak amplitude; WT dark = 7.4 pA, WT light = 22.6 pA and norpA^H43 light = 7.6 pA). D, Intensity–response (R–logI) curve of norpA^H43 mutant flies under low Ca²⁺, 1.5 mm Ca²⁺ and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution. Note that even at extremely high intensities of 1.5 × 10⁷ EP/s under low Ca²⁺ conditions the response amplitude of norpA^H43 photoreceptors was very small. Inset, Intensity–response (logR–logI) relationship (n = 5, mean ± SEM). E, A representative light-induced response to 10⁶ EP/s in norpA^H43 mutant under low Ca²⁺, 1.5 mm Ca²⁺, and under low Ca²⁺ conditions with 1 mm Ca²⁺ in the recording pipette solution.

Figure 8.

Group IIA divalent cations Mg²⁺, Sr²⁺, and Ba²⁺ do not induce dark bumps. A, Whole-cell recordings from WT photoreceptors at total darkness at the various conditions as indicated. Note that only Ca²⁺ induced dark bump activity. B, Histogram plotting the mean bump rate of dark bumps at the different conditions (n = 4, mean ± SEM). C, Light response to 1EP/s intensity. Note that in all cases bumps can be induced by the light stimuli. D, Histogram plotting the mean bump rate of light-induced bumps at the different conditions (n = 4, mean ± SEM).

Figure 9.

Bump kinetics is independent of PLC catalytic activity. A, Whole-cell recordings from WT photoreceptors in total darkness (top trace), light-induced bumps in WT in response to light intensity of 1 EP/s (middle trace) and light-induced bumps in norpA^H43 mutant flies in response to light intensity of 10 EP/s (bottom trace). B, Histogram plotting the time to peak (t_p) distribution of WT dark bumps, WT quantum bumps, and norpA^H43 quantum bumps (n = 118). Inset left, Gaussian fits to the t_p distribution histograms (mean t_p; WT dark = 14.6 ms, WT light = 16.4 ms and norpA^H43 light = 16.8 ms). Inset right, Normalized average of 118 bumps from traces in A (t_p; WT dark = 14.7 ms, WT light = 16.4 ms and norpA^H43 light = 16.1 ms). C, Light response to a brief 50 ms flash at the various group IIA divalent cations solutions as indicated (arrows indicate the t_p of the light response, t_p; Low Ca²⁺ = 189 ms, 1.5 mm Ca²⁺ = 118 ms, 1.5 mm Mg²⁺ = 335 ms, 1.5 mm Sr²⁺ = 141 ms, and 1.5 mm Ba²⁺ = 128 ms). D, Normalized average of 100 bumps at the presence of various group IIA divalent cations solutions as indicated (arrows indicate the t_p of the averaged quantum bump, t_p; Low Ca²⁺ = 71.5 ms, 1.5 mm Ca²⁺ = 16 ms, 1.5 mm Sr²⁺ = 25.7 ms, 1.5 mm Ba²⁺ = 30.6 ms and 1.5 mm Ca²⁺ + 4 mm Mg²⁺ = 17.2 ms).