An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel - PubMed (original) (raw)

. 2018 Apr;14(4):396-404.

doi: 10.1038/s41589-018-0015-6. Epub 2018 Mar 19.

Oleksandra Tiapko 1, Barbora Svobodova 1, Thomas Stockner 2, Toma N Glasnov 3, Wolfgang Schreibmayer 1, Dieter Platzer 1, Gema Guedes de la Cruz 3, Sarah Krenn 1, Romana Schober 4, Niroj Shrestha 1, Rainer Schindl 1, Christoph Romanin 4, Klaus Groschner 5

Affiliations

An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel

Michaela Lichtenegger et al. Nat Chem Biol. 2018 Apr.

Abstract

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

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Figures

Figure 1

Figure 1. Optical control of TRPC3 conductances expressed in HEK293 cells by DAG photoswitches.

a,b, Chemical structures of photoswitchable DAGs:PhoDAG-1 (ref. 18) (1; a) and the newly synthesized OptoDArG (2; b; Supplementary Note). c, Representative time courses of the TRPC3-WT conductances recorded at − 90 mV and + 70 mV during repetitive photoconversion of PhoDAG-1 (400 μM, open circles) and OptoDArG (30 μM, closed circles). UV (365 nm; violet) and blue light (430 nm; blue) irradiation are indicated, with each pulse maintained for 10 s. Time points corresponding to mean values given in d for basal (before illumination; 1), activation (2) and deactivation (3) are indicated. d, Left panel, current densities induced by photoconversion of PhoDAG-1 (400 μM, white) and OptoDArG (30 μM, black) are shown for TRPC3-WT and G652A (at − 90 mV and + 70 mV; mean ± s.e.m. are shown; N = numbers of cells measured, indicated in parentheses) at time points (1,2,3) given in c; two-tailed t-test (normally distributed values) or Mann–Whitney rank sum test (non-normally distributed values) were applied and significant difference at *P < 0.05; ***P < 0.001 are indicated; if no P value is given, comparison with basal (1) current levels is not significant. Values from individual experiments are shown for each of the columns (circles). Right panel, representative current to voltage relations of the PhoDAG-1 and OptoDArG-induced currents as obtained by voltage-ramp protocols measured at blue light irradiation (cis photoconversion) at + 70 mV. Mean current densities ± s.e.m. (N = number of cells measured, indicated in parentheses; corresponding to left panel) for PhoDAG-1 (open circle) and OptoDArG (closed circle); *P = 0.016.

Figure 2

Figure 2. Localization of critical residues within a ‘lipid-gating fenestration’ in TRPC3. Homology model of human TRPC3 based on the Cryo EM structure of TRPV1 (PDB ID 3J9J).

a) Alignment of conserved residues in regions flanking the TRPC1/3/4/5/6/7 and TRPV1 of pore helix. For TRPC3, G652 (red), F618 (magenta) and E630 (green) are highlighted along with the LFW motif (orange). Residue numbering corresponds to the human TRPC3 (hTRPC3) isoform 3. b) Left, top view of four subunits forming the tetrameric TRPC3 channel; G652 is highlighted. Middle, side view of a section showing the poreforming transmembrane helices S5–S6 (residues 570 to 674) of two opposite subunits, with the G652 position indicated relative to the selectivity filter (SF) and the S6 bundle crossing gate (BC). Right, two adjacent chains displaying the subunit interface with a fenestration exposing G652 to the lipid bilayer from the top. c) Space-filling representation of the ‘lipid-gating fenestration’ within the TRPC3 tetramer assembly from an orthoscopic side view.

Figure 3

Figure 3. G652 plays a critical role in TRPC3 gating and activation by DAGs.

a) Left, representative I–V relations of carbachol (CCh)-induced currents (100 μM) through TRPC3-WT (black), G652A (red), or G652L (blue) channels expressed in HEK293 cells. The insert shows the mean reversal potential of illustrated currents. Right, histogram displaying mean current densities of CCh-stimulated (100 μM) HEK293 cells, expressing TRPC3-WT (black), G652A (red), or G652L (blue) channels (right). Mean ± s.e.m. are shown; N = number of cells measured, indicated in parentheses; significant difference at *P < 0.05 and **_P_ < 0.01 are indicated. Values from individual experiments are shown for each of the columns (circles). **b)** Representative time courses for current activation by CCh (100 μM, left panel) and SAG (100 μM, right panel) in TRPC3-WT (black) and G652A (red) mutant channels. **c,d**) Closed time distribution of the nonstimulated (basal, upper left panel) and CCh-stimulated (100 μM; lower left panel) TRPC3-WT (c) and G652A channels (d), expressed in HEK293 cells. Data are derived from single-cell-attached patches at a membrane potential of + 80 mV. The sum of four exponential equations (black) and individual components (red line) with time constants (τ1– τ4) are indicated (**c,d**, left panel). Right panel of **c,d**, statistics of the characteristic opening frequency (f opening) for TRPC3-WT (**c**) and G652A (**d**) mutant channels under basal- and CCh-stimulated conditions as indicated. Mean ± s.e.m. are shown; _N_ = number of cells measured, indicated in parentheses; two tailed _t_-test (normally distributed values) or Mann-Whitney tests (non-normally distributed values) were applied, ns, not significant (_P_ > 0.05). Individual values are shown for each of the data sets (circles).

Figure 4

Figure 4. G652A mutation alters discrimination between DAGs by TRPC3.

a)Bar chart illustrating the current density (at − 90 mV and + 70 mV) of the maximal responses obtained in TRPC3-WT and G652A induced by OAG (100 μM; green), SAG (100 μM; white) and DiC8 (100 μM; gray). Data represent mean ± s.e.m.; N = number of cells measured, indicated in parentheses; two tailed t-test (normally distributed values) or Mann–Whitney tests (non-normally distributed values) were applied, significant differences at *P < 0.05; **_P_ < 0.01 are indicated; ns, not significant (_P_ > 0.05). Values from individual experiments are shown for each of the data sets (circles). b) Representative I–V relations recorded in TRPC3-WT and G652A after application of OAG (100 μM; green) and DiC8 (100 μM; gray)

Figure 5

Figure 5. Photopharmacological determination of the DAG sensitivity of TRPC3-WT and G652A channels.

a) Representative time course of the G652A conductance recorded at − 90 mV and + 70 mV during the repetitive photoconversion of PhoDAG-1 (400 μM, open circles) and OptoDArG (30 μM, closed circles). UV (365 nm; violet) and blue light (430 nm; blue) irradiation is indicated, with each pulse maintained for 10 s. Mean values ± s.e.m. are shown for peak; net-current responses are shown in b. b) Current densities (at − 90 mV and + 70 mV; mean ± s.e.m.; N = number of cells measured, indicated in parentheses) at the maximum, net responses induced by PhoDAG-1 (400 μM, white) and OptoDArG (30 μM, gray) are shown for TRPC3-WT and G652A. Data are mean ± s.e.m.; N = number of cells measured, as indicated; two tailed t-test (non-normally distributed values) or Mann–Whitney test (nonnormally distributed values) were applied and significant differences at *P < 0.05; **_P_ < 0.01; ***_P_ < 0.001 are indicated; if no _P_ level is given, comparison with WT (PhoDAG-1) is not significant (_P_ > 0.05). Values from individual experiments are shown for each of the columns (circles). Individual values are shown for each of the columns (circles). c) Representative responses to increasing light intensity (maximal output power, 670 mW) of TRPC3-WT-(black) and G652A-(red) expressing HEK293 cells induced by photoisomerization of OptoDArG (30 μM). UV (365 nm, violet, pulse held for 2 s) and blue light (430 nm, blue, pulse held for 10 s) are shown. d) Light intensity: current density relation for TRPC3-WT (peak inward current, black; minimum current level during deactivation, gray) and G652A (peak inward current, red; minimum current level during deactivation, deep red) obtained by photoisomerization of OptoDArG (30 μM). Mean ± s.e.m. are shown; N = number of cells measured are indicated; two tailed t-test (non-normally distributed values) or Mann-Whitney tests (non-normally distributed values) were applied; significant difference in comparison to WT indicated at *P < 0.05 and **P < 0.01.

Figure 6

Figure 6. Optical cycling of TRPC3-WT and G652A mutant channels in the presence of OptoDArG (30 μM) occurs with divergent kinetics.

a) Representative traces showing the inward currents induced by OptoDArG (30 μ M) in a whole-cell, gap-free recording (holding potential: − 40 mV, normalized by capacitance) in TRPC3-WT and G652A-expressing HEK293 cells. UV: 365 nm, violet; blue light: 430 nm, blue. TRPC3-WT (black) and G652A (red) responses were induced with 100% intensity of UV, and G652A (blue) was triggered with 50% UV. Exponential fits of current activation for TRPC3-WT (black, 100%), G652A (red, 100%) and G652A (blue, 50%) are shown. b) Exponential fits of the initial phase of current activation for TRPC3-WT (black) and G652A (red). Signal was normalized to peak current. The delay of current activation derived from monoexponential fits is indicated by black arrows. c) Power (n) of power-exponential fitting is shown (f = A*(1-exp(− x/tau))n). Data are mean ± s.e.m.; N = number of cells, indicated in parentheses; two-tailed t-test or Mann-Whitney tests were applied; ns, not significant in comparison to WT. d) Histogram displaying the current activation delay for TRPC3-WT (black) and G652A (red). Data are mean ± s.e.m.; N = number of cells measured, indicated in parentheses; two-tailed t-test or Mann-Whitney test were applied; *P < 0.05. Values from individual experiments are shown for each of the columns (circles).

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