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Mar Drugs
2017 Jun 25;157:. doi: 10.3390/md15070198.
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Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes.
Flores PL
,
Rodríguez E
,
Zapata E
,
Carbó R
,
Farías JM
,
Martínez M
.
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Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.
Figure 1. Currents induced by maitotoxin (MTX) in control oocytes. (A) Temporal course of currents induced by 10 pM MTX obtained by the two-electrode voltage-clamp technique (TEVC) and with a hyperpolarizing protocol of pulses at three membrane voltages (−160, −145, and −130 mV). MTX was added at the point indicated by the red arrow. The MTX-induced currents showed a maximal effect in about 15 min. MTX was washed out by perfusion with Ringer solution, and complete recovery was reached after 45 min. Representative recordings traced before adding MTX, at the maximal current level, and at the end of a continuous washing are shown as inserts. (B) Current–voltage (I–V) relationships for the peak of currents obtained for all three conditions (control, MTX, and recovery) are shown (n = 3). The symbols represent the means, and the bars represent the standard error of the mean (s.e.m.).
Figure 2. Effects of MTX in oocytes injected with transient receptor potential channels 1 and 4 (TRPC1 and TRPC4) siRNAs. (A) Temporal course of the MTX effects in oocytes injected with TRPC1 and TRPC4 siRNAs at day three after injection. The symbols represent the mean (±SD) of the maximal current obtained at three membrane voltages (−160, −145, and −130 mV) in three independent experiments. In oocytes injected with TRPC1 siRNA, MTX did not induce evident currents at 10 and 50 pM. The mean of currents from oocytes injected with TRPC4 siRNA was similar to non-injected oocytes. (B) Representative recording traces for each condition. The current traces from oocytes injected with TRPC1 siRNA were obtained at points a, b, c (in red), as well as the current traces from oocytes injected with TRPC4 siRNA in points a’, b’, and c’ (in bue) are shown.
Figure 3. TRPC1 and TRPC4 expression in injected oocytes. (A) TRPC1 mRNA levels at different times of incubation after TRPC1 siRNA injection; (B) and (C) show the protein expression levels for TRPC1 and TRPC4, respectively. Values are the results of the means ± SD, n = 3; each experiment was formed with at least 15 oocytes. A one-way analysis of variance (ANOVA) was performed, followed by a Mann–Whitney test.
Figure 4. MTX effect on N,N′-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA-AM (BAPTA-AM)-incubated oocytes. (A) Temporal course of MTX effect (10 and 50 pM) on pre-incubated oocytes with 200 nM BAPTA-AM and on non-incubated oocytes. The maximal current level (mean ± SD; n = 4) at three membrane voltages are shown (−160, −145, and −130 mV) for incubated and non-incubated oocytes. (B) Representative current traces of the MTX effect extracted from the temporal course experiments are indicated for each condition. (C) The current–voltage relationships for the maximal current (mean ± s.e.m.) for incubated and non-incubated cells are shown (n = 4).
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