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Science 338 (6104): 260-263

Copyright © 2012 by the American Association for the Advancement of Science

Photomechanical Responses in Drosophila Photoreceptors

Roger C. Hardie*, and Kristian Franze

Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK.

Figure 1
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Fig. 1. AFM measurements of photomechanical responses. (A) An AFM cantilever contacts distal tips of ommatidia in an excised Drosophila retina. (B) An ommatidium, containing photoreceptors (orange) and pigment cells (red). Elements of the phototransduction cascade are contained within microvillar rhabdomeres (two shown in longitudinal section, seven in cross section), which are rodlike stacks ~80 μm in length containing ~30,000 microvilli. Right: Electron micrograph cross section of a rhabdomere (scale bar, 1 μm), showing tubular microvilli, each ~50 nm in diameter, with lumen in diffusional continuity with the cell body. (C) Phototransduction cascade. Rhodopsin (R) is photoisomerized to metarhodopsin (M*), which catalyzes release of the Gq protein α subunit to activate PLC. PLC hydrolyzes PIP2 (red), leaving DAG (green) in the membrane. Ca2+ influx via TRP channels inhibits PLC. (D) Lower traces: AFM measurements of contractions (cantilever z-position) in a wild-type retina in response to 5-ms flashes, with intensity increased from ~200 to 8000 effectively absorbed photons per photoreceptor. Blue traces: Whole-cell current-clamped voltage responses to the same stimuli recorded from a dissociated photoreceptor cell. (E) Contractions evoked by 5-ms flashes covering the full intensity range (~200 to 106 photons) in a wild-type retina. (F) Same on faster time base. (G) Response versus intensity (R/I) functions of contractions (nm) from wild-type retinae (mean ± SEM, N = 13), and peak voltage (mV) recorded from dissociated photoreceptors (blue; means ± SEM, N = 6). (H) Responses to flashes (~5 x 104 photons) in trpl mutant before and after (red) channel block by 50 μM La3+ and 10 μM ruthenium red (RR), which prevents inhibition of PLC by Ca2+ influx. Blue trace: Lack of response in norpAP24 (PLC mutant) despite using flashes of higher intensity (~2 x 105 photons; N = 3). (I) R/I function of contractions from trpl retinae before and after (red) channel block by La3+ and RR (means ± SEM, N = 5). For intensity calibration, see fig. S2.


Figure 2
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Fig. 2. Light-induced modulation of gramicidin channels. (A) Whole-cell recordings from trpl;trp mutant photoreceptor cell lacking all native light-sensitive channels. Perfusion with gramicidin induced a constitutive inward current. (B) Flashes of increasing intensity (5 x 103 to 4 x 105 effective photons), each 1 s (denoted by bar at upper left), up-regulated the current. (C) Averaged responses (±SEM) to 100-ms flashes containing 1.3 x 104 (middle trace, N = 4) and 3.7 x 104 (lower trace, N = 10) effective photons (data pooled from trpl;trp and trpl mutants recorded in the presence of La3+ and RR). The same flashes delivered before gramicidin application (controls) induced residual, noise-free transient currents of uncertain origin. (D) R/I function (after subtracting control responses measured before gramicidin perfusion) expressed as a fraction of the steady-state gramicidin current I/ISS (means ± SEM, N = 4). Dotted curve: R/I function of contractions measured by AFM in trpl mutant in the presence of La3+ and RR, replotted from Fig. 1I.


Figure 3
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Fig. 3. Modulation of light-sensitive channels by osmotic pressure. (A to D) Whole-cell voltage-clamped responses to 1-ms flashes (~50 effective photons) in control bath (300 mOsm) were reversibly increased by perfusion with 200 mOsm solution and suppressed by 400 mOsm in wild-type (A), trpl (B), and trp (C) mutants as well as in wild-type photoreceptors recorded in Ca2+-free solutions (D). (E) Response amplitudes (I/I300) after hyperosmotic (400 mOsm) and hypo-osmotic (200 mOsm) challenges normalized to control responses in 300 mOsm bath. Data are means ± SEM; N = 4 to 8 cells. All conditions plotted were significantly increased (200 mOsm) or decreased (400 mOsm) relative to control responses from the same cells [P < 0.005; analysis of variance (ANOVA) followed by posttest for trend]. (F) Spontaneous TRP channel activity (from trpl mutant) after several minutes of recording, using pipettes lacking nucleotide additives. Perfusion with 400 or 200 mOsm (bar) reversibly suppressed and facilitated this "rundown current" (RDC). (G) Channel noise resolved on a faster time base, plus trace recorded in dark before onset of RDC. (H) Left: Amplitude of steady-state RDC normalized to value at 300 mOsm. Right: Effective single-channel conductance ({gamma}) estimated by variance/mean ratio (means ± SEM, N = 7). Although macroscopic RDC was substantially modulated (P < 0.001; ANOVA, posttest for trend), single-channel conductance was not significantly affected by osmotic manipulation (P > 0.2).


Figure 4
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Fig. 4. Cationic amphipaths inhibit the light response. (A) Response to 1-ms flashes in trp mutants in the presence of procaine (PROC), imipramine (IMP), trifluoperazine (TFP), and chlorpromazine (CPZ). Control responses before (turquoise) and after washout (blue) are superimposed. (B) Dose response functions (means ± SEM: PROC, N = 4; IMP, N = 3; TFP, N = 3; CPZ, N = 6) fitted by inverse Hill curves (slope constrained at n = 2), based on raw values (IC50: PROC, 3.1 mM; IMP, 27 μM; TFP, 4.9 μM; CPZ, 4.5 μM). (C) Same as in (B) after correction for pKa and octanol partition coefficients, reflecting predicted concentration in the lipid membrane (IC50: PROC, 2.7 mM; IMP, 7 mM; TFP, 3.4 mM; CPZ, 2.3 mM).


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