BTDAzo: A Photoswitchable TRPC5 Channel Activator

Abstract

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca²⁺ -permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca²⁺ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo's ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

Keywords: Agonists; Cation Channels; Ligand Design; Photopharmacology; Prolactin Signaling.

Figure 1

Design (see Supporting Information for high‐resolution vectorial copies of all main text Figures). a) Known TRPC5 agonists BTD, Englerin A, AM237. b) BTDAzo, the best photoswitchable agonist of the series btda1–btda7 (cf. Figure S1).

Scheme 1

Synthesis of BTDAzo (btda3).

Figure 2

Photocharacterisation. a) E⇄Z isomerisations of BTDAzo. b) Z : E ratios at PSS depending on environment. c) PSS spectra of BTDAzo in H₂O/DMSO. d) Spectra of pure E‐ and Z‐ BTDAzo (inline HPLC detection). e) BTDAzo can be reversibly photoswitched between PSS states.

Figure 3

Cellular TRPC5 photoswitching with BTDAzo (see Supporting Information for high‐resolution vectorial copies of all main text Figures). a), b) Reversible Ca²⁺ influx modulation with BTDAzo under cycles of 365/447 nm illumination, and peak amplitudes (mostly‐Z‐ BTDAzo), as detected in Fluo‐4‐loaded HEK_mTRPC5‐CFP cell suspensions by fluorescence plate imager (values in b are means±S.E., averaged over the time periods illustrated between dotted vertical lines in panel a). For additional information see Figure S5. c)–f) Microfluorometric single cell analysis (60 grey traces: single cells; black trace: averaged signal) of TRPC5 activation measured with a monochromator‐equipped Xenon light source. c), d) Cellular action spectrum for stimulation of Ca²⁺ influx into single HEK_mTRPC5‐CFP cells by E→Z BTDAzo isomerisation. e), f) Cellular action spectrum for preventing Ca²⁺ influx, by Z→E BTDAzo isomerisation at various wavelengths immediately after E→Z isomerisations. g)–i) Electrophysiological whole‐cell recordings of TRPC5 currents in voltage clamp (V _h=‐80 mV) mode. g) Ionic currents in a TRPC5‐expressing HEK293 cell during 60 consecutive cycles of 360/440 nm illumination in the presence of 10 μM BTDAzo. Dotted line shows zero current level (half of each time course shown). h) Ionic currents in TRPC5/TRPC1‐co‐expressing HEK293 cell, as in panel g. i) Magnification of single on‐off cycles taken from the traces shown in (g), (h) as indicated by the coloured boxes. j)–k) I/V curves of whole cell currents in voltage‐clamped TRPC5‐ or TRPC5/TRPC1‐expressing cells exposed to 360 nm and 440 nm light with 10 μM BTDazo in the bath solution. Note the distinct shape of the I/V curve in panel k, with smaller inward current component. All data from a minimum of 3 independent experiments, each run in technical duplicates.

Figure 4

BTDAzo photocontrols endogenous TRPC5‐dependent Ca²⁺ responses in mouse brain (see also Figure S9). a) Cascade model for TRPC5‐dependent cation channel activation in Th+ neurons of the hypothalamic arcuate nucleus (ARC) after stimulation of the prolactin receptor (PrlR). b) Cartoon of the coronal brain slice, and region of the ARC (blue). Dashed box indicates the ARC region in (c). c) Pseudocoloured image of Th+ neurons expressing GCaMP6f in a mouse brain slice. Inset: Zoom on a GCaMP6f neuron from the dorsomedial region of the ARC. (3 V, third ventricle; ME, median eminence). d)–g) Original traces of spontaneous Ca²⁺ responses in Th+ neurons of Th‐GCaMP6f mice, stimulated with prolactin (d), BTD (e), or BTDAzo followed by E→Z isomerisation using 355 nm UV laser stimulation (f: total UV exposure time, 14.1 ms; response delay, 48 s). f), g) Panel (g) shows data for the TRPC5‐deficient Th+ neuron of Th‐GCaMP6f‐ΔTrpc5 mouse (total UV exposure time, 14.5 ms). h) Area under the curve (AUC) of Ca²⁺ signals when applying BTDAzo under 488 or 355 nm in wildtype or Trpc5 deficient Th+ neurons, compared with wildtype cosolvent only (Con) or BTD controls. Two‐way ANOVA: F(3, 158)=3.011, p<0.05. i) Wildtype or knockout AUCs normalized to their controls (cosolvent or 488 nm stimulation of BTDAzo) show prolactin, BTD, and BTDAzo after 355 nm photoswitching stimulate TRPC5‐dependent Ca²⁺ increases. Kruskal‐Wallis ANOVA: χ²(9)=95.24, p<0.00001. [h, i): number of cells is indicated in parentheses above each bar; for full description of material methods and statistics, see Supporting Information.]