High-Sensitivity Fluorometry to Resolve Ion Channel Conformational Dynamics  Matthias Wulf, Stephan Alexander Pless  Cell Reports  Volume 22, Issue 6, Pages 1615-1626 (February 2018) DOI: 10.1016/j.celrep.2018.01.029 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 General Setup and Procedure (A) Schematic illustration of hsPCF setup. Fluorescently labeled receptors in a membrane patch are illuminated via a light-emitting diode (LED) light source through a digital mirror device (DMD), directing the light beam with higher spatial resolution than conventional illumination (compare green light beam with yellow light beam in inset). Ligands are applied via a custom-built perfusion tool actuated by a piezo element. Emitted light is detected with an electron multiplying charge-coupled device (EMCCD) camera or an avalanche photo diode (APD) after passing a physical mask. Standard patch-clamp configuration is used to record ionic currents. (B) Workflow for hsPCF recordings. (I) A patch is pulled from a fluorescently labeled Xenopus laevis oocyte expressing the protein of interest. (II) The patch electrode is placed in front of the perfusion tool in control solution. (III) The background around the glass tip is covered with a physical mask (black frame) and the DMD is focused on the patch (green square) for active illumination. (IV) Averaged pixel intensity from a selected region of interest (indicated by red circle) is analyzed. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Increased Signals through the Use of DMD and Physical Mask (A) Representative current (black) and unfiltered fluorescence (red) hsPCFoo recordings from a single sweep of GlyR α1 Q67C with EMCCD at 1.5 kHz (left panel) and averaged ΔF/F values comparing VCF (white bar, n = 8) and hsPCFoo (red bar, n = 17). The illustrated ROIs (dotted circles) in the image inset show the analyzed regions to assess fluorescence intensities of the patch (P, red) and of the background (B, gray). A cartoon of a recording pipette illustrates the orientation of a labeled (red dot) membrane protein in this and all subsequent figures. Statistical significance was assessed with unpaired t test; ∗∗∗p < 0.001. (B) Comparison of fluorescence change in the patch (ΔFP, red bars), the background fluorescence (FB, gray bars) and fluorescence changes in the background (ΔFB, black bars) in hsPCFoo recordings with DMD and physical mask either both off or on or individually on (all four combinations were recorded from the same patches). Groups were compared with repeated-measure one-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. All bars depict mean ± SEM. See also Figure S1 and Tables S1 and S2. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Speed and Sensitivity: Resolving Conformational Changes in Loop D of GlyR α1 (A) Unfiltered fluorescence trace from a single sweep recorded with APD at 10 kHz from GlyR α1 Q67C. Inset shows expanded timescale around signal onset upon 10 mM glycine application. (B) Representative traces showing current (black) and fluorescence (red) changes in response to 200 μM glycine application (left panel), PTX (200 μM) alone (middle panel), and co-application of glycine and PTX (right panel). Representative single exponential fits are shown in gray for current and black for fluorescence recordings. Note the scale bars for current size, indicating very small channel populations (<100 channels). (C) Kinetic analysis of time course for current (black bars) and fluorescence (red bars) changes in response to application of glycine alone and co-application with PTX. τon values are subdivided in initial (i) glycine application, glycine application after PTX removal (r), and τoff values during PTX and control (ctrl) solution. See also right panel in (B). Groups were compared with repeated-measure one-way ANOVA; ∗p < 0.05, ∗∗p < 0.01. All bars show mean ± SEM. See also Figure S2 and Table S3. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Resolving Voltage-Sensor Kinetics of VGICs (A) Shaker A359C ΔF/F signals from hsPCF recordings (red bars) from outside-out (oo, n = 9) and inside-out (io, n = 25) patch configurations compared with VCF recordings (white bar, n = 10) for a voltage step from −120 mV to +20 mV. (B) hsPCFio recordings of Shaker A359C for indicated voltage protocol with intracellular KCl (gray), TEA (orange, top panel), or CsCl (cyan, bottom panel). Current and fluorescence traces are shown in black and red, respectively. Representative single exponential fits (dotted black line) for τOn and τOff are superimposed with fluorescence traces. (C) Activation (τon, white bar) and deactivation (τoff, black bar) kinetics of the fluorescence signal with intracellular ions color-coded as in (B). While τon kinetics were unaffected by CsCl and TEA, τoff kinetics were significantly faster in CsCl (lower panel, n = 14) but significantly slower in TEA (top panel, n = 8). τoff and τon groups were compared with an repeated-measure one-way ANOVA; ∗p < 0.05, ∗∗∗p < 0.001. All bars show mean ± SEM. See also Figure S3 and Table S4. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 hsPCF Is Compatible with Fluorescent Noncanonical Amino Acids (A) Structure of ANAP methyl ester. (B) Representative current (black) and unfiltered fluorescence (red) hsPCFoo trace from a single recording of GlyR α1 A52TAG at 1 kHz with EMCCD. The oscillation in the fluorescence trace following the glycine application is likely caused by mechanical movement of the perfusion tool through the piezo actuator. (C) Comparison of averaged ΔF/F values between VCF (white bar, n = 8) and hsPCFoo (red bar, n = 14) from GlyR α1 A52TAG with unpaired t test; ∗∗∗p < 0.001. (D) Contribution of DMD and physical mask on fluorescence signal for GlyR α1 A52TAG (for details, see Figure 2B). All data are shown as mean ± SEM. See also Figure S5 and Tables S5 and S6. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 Following Conformational Changes in the ASIC1a Pore (A) Representative current (black) and fluorescence (red) trace recorded from ASIC1a F440TAG with an extracellular pH change from 7.4 to 5.5 in an outside-out patch (left panel) and average fluorescence change recorded upon extracellular acidification (right panel, n = 9). (B) Representative current and fluorescence trace from ASIC1a F440TAG construct for intracellular application of TEA in an inside-out patch (left panel) and bar diagram depicting average fluorescence changes with intracellular application of TEA (right panel, n = 7). All data are shown as mean ± SEM. Cell Reports 2018 22, 1615-1626DOI: (10.1016/j.celrep.2018.01.029) Copyright © 2018 The Author(s) Terms and Conditions