Lipid-independent control of endothelial and neuronal TRPC3 channels by light

Abstract

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca²⁺ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca²⁺ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

Scheme 1. Synthesis of selected analogues of GSK1702934A (GSK), designated as BI-1, BI-2, PI, and the corresponding photoresponsive derivatives designated as OptoBI-1, OptoBI-2 and OptoPI. Yield is given in %.

Fig. 1. (a) Chemical structures of GSK1702934A (GSK) and GSK derivatives (BI-1, BI-2 and PI). (b) Current to voltage relations of the net conductances (I_max – I_basal) induced by GSK, BI-1, BI-2 and PI (10 μM each) obtained in TRPC3-transfected HEK293 with voltage-ramp protocols. (c) Representative time courses of the TRPC3 conductances recorded at –90 mV and +70 mV during administration of channel activators (GSK, BI-1, BI-2 and PI as indicated) at 10 μM concentrations. (d) Current density of net, maximum responses obtained at –90 mV and +70 mV (mean ± SEM). Statistical significance was tested by two tailed t-test (normally distributed values) or Mann–Whitney tests (non-normally distributed values). ns = not significant (p > 0.05); N cells: GSK = 9; BI-1 = 6; BI-2 = 7; PI = 5.

Fig. 2. (a) Chemical structures of photoswitchable GSK derivatives: OptoBI-1, OptoBI-2, OptoPI. (b) Representative time courses of the TRPC3 conductances recorded at –90 mV and +70 mV during repetitive photoconversion of OptoBI-1, OptoBI-2 and OptoPI (10 μM). Cells were kept in dark prior to light application, following by cycling of UV (365 nm; violet) and blue light (430 nm; blue) illuminations, applied for 10 s each (indicated). (c) Representative net I–V relations (I_max – I_basal) for OptoBI-1, OptoBI-2 and OptoPI-induced (10 μM) obtained in TRPC3-transfected HEK293 cells with voltage-ramp protocols. (d) Dependency of inward current densities (at –90 mV), induced by photoconversion of OptoBI-1 at increasing concentrations. Values (mean ± SEM) are given for currents before illumination (squares; basal/trans; N/cells = 8), during UV-induced cis isomerization (triangles; cis; N/cells = 8) and upon deactivation cycling by blue light (circles; trans; N/cells = 8). (e) Time courses of Ca²⁺-sensitive R-GECO fluorescence during OptoBI-1 (10 μM) photoconversion in TRPC3 plus R-GECO (red and black trace) and R-GECO only (blue trace) expressing in HEK293. Illumination (365 nm; 10 s and 430 nm; 30 s) performed before fluorescence recording, is indicated by arrows (N of cells: YFP-TRPC3 and OptoBI-1 = 27; YFP-TRPC3 and DMSO = 5; HEK293 WT and OptoBI-1 = 14). (f) Fluorescence levels before and after UV light-induced changes in cells co-transfected with TRPC3 and R-GECO in presence of DMSO (control) or cis-photoconversion of OptoBI-1 and OptoPI (10 μM). Mean ± SEM, ns = not significant (p > 0.05), *p < 0.05, statistical significance was tested by two tailed t-test; N = number of cells as indicated in (e); individual values are included (circles).

Fig. 3. (a) Time course of Ca²⁺ sensitive Fluo-4 fluorescence in EA.hy926 non-transfected WT cells (control (DMSO) N cells used = 23; OptoBI-1, 60 μM, N = 23) or cells transfected with CFP-TRPC3 (OptoBI-1, 60 μM; N = 21) in 2 mM Ca²⁺ buffer upon cis-photoisomerization with UV light (365 nm; 15 s). (b) Change in bleaching-corrected Fluo-4 fluorescence evoked by cis-OptoBI-1 (60 μM) in EA.hy926 cells (WT, N = 23; CFP-TRPC3 transfected, N = 21 and WT in Ca²⁺-free, N = 10). Control cells (N = 23) were incubated with DMSO in 2 mM Ca²⁺ buffer; statistical significance was tested by two tailed t-test (normally distributed values) or Wilcoxon signed rank test (non-normally distributed values); ns = not significant (p > 0.05), ***p < 0.001. (c) Representative AP firing in primary hippocampal neurons (WT) induced by a short (5 s) current injection. The first current injection was performed in dark (trans conformation; control), following by cis isomerization with UV (365 nm) and subsequent reversal to trans conformation with blue (430 nm) light. (d) Mean AP count/pulse in dark, UV and blue light-stimulated in current-clamped WT controls (DMSO) and incubated with OptoBI-1 (20 μM) WT and TRPC1–7 KO neurons. Bar graphs show mean ± SEM from at least 3 different preparations; ns = not significant (p > 0.05), *p < 0.05; **p < 0.01; statistical significance was tested by two tailed t-test (normally distributed values) or Mann–Whitney tests (non-normally distributed values). Individual values are indicated (circles).