Tonantzitlolone is a nanomolar potency activator of transient receptor potential canonical 1/4/5 channels

Abstract

Background and purpose: The diterpene ester tonantzitlolone (TZL) is a natural product, which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-englerin A, and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the 1, 4 and 5 type (TRPC1/4/5 channels).

Experimental approach: The effects of TZL on renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK293 cells overexpressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2, or CHO cells overexpressing TRPV4 were studied by determining changes in intracellular Ca²⁺ , or whole-cell or excised membrane patch-clamp electrophysiology.

Key results: TZL induced an elevation of intracellular Ca²⁺ in A498 cells, similar to that evoked by englerin A. TZL activated overexpressed channels with EC₅₀ values of 123 nM (TRPC4), 83 nM (TRPC5), 140 nM (TRPC4-TRPC1) and 61 nM (TRPC5-TRPC1). These effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca²⁺ entry or overexpressed TRPC3, TRPV4 or TRPM2 channels in HEK 293 cells.

Conclusions and implications: TZL is a novel potent agonist for TRPC1/4/5 channels, which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell.

Figure 1

Chemical structure of TZL.

Figure 2

TZL causes an elevation in intracellular Ca²⁺ in A498 cells. (A) Representative intracellular Ca²⁺ measurement data from a single 96‐well plate showing the effect of the vehicle control, 100 nM TZL or 100 nM (‐)‐englerin A (EA) (n = 1 independent experiment and N = 3 replicate wells each). (B) For experiments of the type shown in (A), mean ± SEM TZL data normalized to the amplitude of englerin A response (n = 6 independent experiments). Responses were measured 450–480 s after starting the application of the compound. ^* P < 0.05.

Figure 3

TZL induces an elevation in intracellular Ca²⁺ in TRPC1/4‐, TRPC4‐, TRPC5‐ and TRPC1/5‐overexpressing HEK293 cells. (A) Representative traces from one independent experiment on TRPC1/4 cells exposed to increasing concentrations of TZL (3–3000 nM) (N = 4 replicate wells for each trace). (B) For experiments of the type shown in (A), mean ± SEM data fitted with the Hill equation to determine the EC₅₀ (n/N = 6/24, i.e. n = 6 independent experiments and N = 4 replicates per independent experiment). (C) Representative traces from TRPC4 cells exposed to increasing concentrations of TZL (3–1000 nM) (N = 4 each). (D) For experiments of the type shown in (C), mean ± SEM data fitted with the Hill equation to determine the EC₅₀ (n/N = 6/18). (E) Representative traces from TRPC5 cells exposed to increasing concentrations of TZL (3–3000 nM) (N = 4 each). (F) For experiments of the type shown in (E), mean ± SEM data fitted with the Hill equation to determine the EC₅₀ (n/N = 6/24). (G) Representative traces from TRPC1/5 cells exposed to increasing concentrations of TZL (3–1000 nM) (N = 4 each). (H) For experiments of the type shown in (G), mean ± SEM data fitted with the Hill equation to determine the EC₅₀ (n/N = 6/24).

Figure 4

TZL‐activated TRPC5 responses are inhibited by Pico145 and similarly shown by whole‐cell patch‐clamp recording. Data are for HEK293 cells overexpressing TRPC5. (A) Representative traces for one independent experiment on intracellular Ca²⁺ showing responses to 3 μM TZL in the absence and presence of 30 nM Pico145 (N = 4 replicate wells each). (B) For experiments of the type shown in (A), mean ± SEM data (n/N = 6/24). ^* P < 0.05. (C) Representative whole‐cell patch‐clamp recording showing the effect of bath‐applied 1 μM TZL and 30 nM Pico145 as indicated by horizontal bars. Currents were sampled at −100 and +100 mV during ramp changes in voltage from −100 to +100 mV. (D) For experiments of the type shown in (C), example current–voltage relationships (IVs) for before TZL application (No TZL), at the maximum response to TZL (TZL), after wash‐out of TZL (Wash) and after Pico145 was applied in addition to TZL (+Pico145). Representative of six independent experiments. (E) Representative whole‐cell patch‐clamp data showing currents sampled at −100 and +100 mV during ramp changes in voltages from −100 to +100 mV. (F) For experiments of the type shown in (E), example IVs for before TZL application (No TZL) and in response to increasing concentrations of TZL (10–1000 nM). Representative of five independent experiments. (G) For experiments of the type shown in (E, F), mean ± SEM concentration–response data for +100 mV (G) and −100 mV (H) fitted with the Hill equation to yield EC₅₀ values and slopes of 76.5 ± 9.2 nM and 0.89 (G) and 64.5 ± 7.3 nM and 0.82 (H).

Figure 5

Bath‐applied TZL activates TRPC5 in excised outside‐out but not inside‐out patches. Data were from HEK293 cells overexpressing TRPC5. (A, B) Inside‐out patch recording in which 1 μM TZL was bath‐applied as indicated by the horizontal bar, showing a typical time‐series recording (A) and IVs from this recording (B). Representative of seven independent recordings (i.e. n = 7). (C, D) Outside‐out patch recording in which 1 μM TZL and 30 nM Pico145 were bath‐applied as indicated by the horizontal bars, showing a typical time‐series recording (C) and IVs from this recording (D). Representative of six independent recordings (i.e. n = 6).

Figure 6

TZL does not activate native store‐operated Ca²⁺ entry channels or overexpressed TRPC3, TRPV4 or TRPM2 channels. Intracellular Ca²⁺ measurements were made from cell lines stably expressing TRPC3 (A), TRPV4 (B) or TRPM2 (C), and shown are representative independent experiments from n = 6 each. Data are presented as mean ± SEM. (A) TRPC3 channels were activated by 100 μM 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) but not 1 μM TZL (N = 4 replicate wells each). (B) TRPV4 channels were activated by 5 μM 4α‐phorbol 12,13‐didecanoate (4α‐PDD) but not 1 μM TZL (N = 5 replicate wells each). (C) TRPM2 channels were activated by 1 mM hydrogen peroxide (H₂O₂, black) but not 1 μM TZL (red) (N = 5 replicate wells each).