Uchida K et al. (2019), Identification and classification of a new TRPM...

XB-ART-55897

J Physiol Sci 2019 Jul 01;694:623-634. doi: 10.1007/s12576-019-00677-6.

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Identification and classification of a new TRPM3 variant (γ subtype).

Uchida K , Fukuta N , Yamazaki J , Tominaga M .

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TRPM3 is a non-selective cation channel that is activated by neural steroids such as pregnenolone sulfate, nifedipine, and clotrimazole. Despite the number of TRPM3 variants, few reports have described functional analyses of these different TRPM3 types. Here we identified a new TRPM variant from mouse dorsal root ganglion, termed TRPM3γ3. We classified TRPM3γ3 and another known variant (variant 6) into the γ subtype, and analyzed the TRPM3γ variants. mRNA expression of TRPM3γ was higher than that of TRPM3α variants in the mouse dorsal root ganglion. In Ca2+-imaging of HEK293 cells expressing either the TRPM3γ variants or TRPM3α2, increases in cytosolic Ca2+ concentrations ([Ca2+]i) induced by pregnenolone sulfate or nifedipine were smaller in cells expressing the TRPM3γ variants compared to those expressing TRPM3α2. On the other hand, co-expression of TRPM3γ variants had no effect on [Ca2+]i increases induced by pregnenolone sulfate or nifedipine treatment of HEK293 cells expressing TRPM3α2. In Xenopus oocytes, small responses of TRPM3γ variants to chemical agonists compared to TRPM3α2 were also observed. Interestingly, Xenopus oocytes expressing TRPM3α2 displayed heat-evoked currents with clear thresholds of about 40 °C that were larger than those evoked in oocytes expressing TRPM3γ variants. Overall, these findings indicate that TRPM3γ variants have low channel activity compared to TRPM3α.

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Species referenced: Xenopus
Genes referenced: neff1 trpm3

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	Fig. 1 TRPM3 variant topology. The uppermost line indicates the membrane topology model of TRPM3. Black bars for each variant indicate coding regions. Yellow allows indicate primers for specific amplification of each variant. Primer sequences are shown in Table 1. Numbered exons indicate regions that are deleted in variants. ICF indispensable for channel function region, TM transmembrane domain, CC coiled-coil domain (color figure online)
	Fig. 2 Expression levels of TRPM3α and TRPM3γ mRNA in mouse dorsal root ganglion. a Design of primer sets for quantifying the TRPM3 variants. Different primer pairs and their location relative to the TRPM3 mRNA are shown as arrows. Primer sequences are shown in Table 1. b mRNA expressions of TRPM3 variants (α+β+γ, α, and γ) in mouse dorsal root ganglion by quantitative real-time RT-PCR analysis. Y-axis: Copy number of mRNAs per 1 μl mRNA sample. Each bar represents the mean + SEM of six mice. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. **P < 0.01 versus TRPM3α+β+γ, ##P < 0.01 versus TRPM3α
	Fig. 3 [Ca2+]i increases induced by pregnenolone sulfate or nifedipine treatment of HEK293T cells expressing mouse TRPM3γ or TRPM3α variants. a, b Average changes in [Ca2+]i induced by pregnenolone sulfate (PS, a) or nifedipine (b) treatment of HEK293T cells expressing TRPM3α2 or TRPM3γ2. Cell viability was confirmed by application of 5 μM ionomycin (Iono). The y-axis shows the fura-2 ratio of 340 nm/380 nm. Each symbol represents mean ± SEM of 51–108 cells. c, d Effect of 3, 10, or 50 μM PS (c) and 10, 30, or 100 μM nifedipine (d) on HEK293T cells expressing mouse TRPM3α2, TRPM3α3, TRPM3γ2, or TRPM3γ3. Mock shows results for vector-transfected HEK293T cells. Y-axis: the Δ ratio normalized to ionomycin responses. Each column represents the mean + SEM of 80–280 cells. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. **P < 0.01 versus TRPM3α2, ##P < 0.01 versus TRPM3α3
	Fig. 4 Effects of TRPM3γ2 or TRPM3γ3 co-expression with TRPM3α2 on [Ca2+]i changes induced by pregnenolone sulfate or nifedipine. [Ca2+]i increases induced by pregnenolone sulfate (PS, a) or nifedipine (b) treatment of HEK293T cells expressing mouse TRPM3α2 without or with TRPM3γ2 or TRPM3γ3. Y-axis: the Δ ratio normalized to ionomycin responses. Each column represents the mean + SEM of 95–171 cells
	Fig. 5 Currents activated by pregnenolone sulfate or nifedipine in Xenopus oocytes expressing mouse TRPM3α2, TRPM3γ2, or TRPM3γ3. a–d Representative traces of endogenous (mock, a), TRPM3α2 (b), TRPM3γ2 (c), or TRPM3γ3 (d) currents activated by treatment of Xenopus oocytes with 1–300 μM pregnenolone sulfate. The membrane potential was held at − 60 mV. e Representative current–voltage curves of the currents by treatment of distilled water-injected Xenopus oocytes (mock) or Xenopus oocytes expressing TRPM3α2, TRPM3γ2, or TRPM3γ2 with 1, 10, and 100 μM pregnenolone sulfate (PS). f Dose–response profiles of currents in Xenopus oocytes expressing TRPM3α2, TRPM3γ2, or TRPM3γ3 activated by pregnenolone sulfate. Mock indicates the currents by pregnenolone sulfate in distilled water-injected Xenopus oocytes. Hill coefficients were 1.3 ± 0.1 (TRPM3α2), 1.4 ± 0.2 (TRPM3γ2) and 1.5 ± 0.2 (TRPM3γ3). Each symbol represents the mean ± SEM of 9–11 oocytes. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. P < 0.05, P < 0.01, versus DW, #P < 0.05, ##P < 0.01, versus TRPM3α2. g–i Representative traces of endogenous (mock, g), TRPM3α2 (h) or TRPM3γ2 (i) currents activated by 100 μM nifedipine in Xenopus oocytes. The membrane potential was held at − 60 mV. Pregnenolone sulfate (PS, 100 μM) was applied in the end. j Representative current–voltage curves of the currents by 100 μM nifedipine in distilled water-injected Xenopus oocytes (mock) or Xenopus oocytes expressing TRPM3α2, TRPM3γ2, or TRPM3γ3 with 100 μM nifedipine (Nif). k Comparison of peak nifedipine (100 μM)-evoked currents in distilled water-injected Xenopus oocytes (mock), and Xenopus oocytes expressing TRPM3α2, TRPM3γ2, or TRPM3γ3. Each column represents the mean ± SEM of 5–10 oocytes. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t-test with Bonferroni correction. *P < 0.01, versus DW, ##P < 0.01, versus TRPM3α2
	Fig. 6 Temperature-activated currents in Xenopus oocytes expressing mouse TRPM3α2. a, d Representative traces of TRPM3α2 currents activated by rapid (a) or slow (d) temperature changes up to 45 °C in Xenopus oocytes. Pregnenolone sulfate (PS, 100 μM) was applied after the temperature stimulus and the membrane potential was held at − 60 mV. b, e Temperature–current profiles from the traces in a and d, respectively. The x- and y-axes show temperature (°C) and current (μA), respectively. c, f Arrhenius plots from the traces in a and d, respectively. The lower and upper x-axes show 1000/temperature (K) and temperature (°C), respectively, whereas the y-axis shows the common logarithmic plot of current size. Q10 values were calculated from the approximate lines shown in black. The intersection of the two linear-fitted lines was defined as a temperature threshold as shown by the dashed line. g Comparison of the temperature thresholds for rapid and slow heat-evoked TRPM3α2 activation. Each column represents the mean ± SEM of 6–8 oocytes. Statistical significance was assessed using Student’s t test
	Fig. 7 Temperature-activated currents in Xenopus oocytes expressing mouse TRPM3γ2 or TRPM3γ3. a, b Representative traces of TRPM3γ2 (a) or TRPM3γ3 (b) currents activated by rapid temperature changes up to 45 °C in Xenopus oocytes. c Comparison of sizes of heat-evoked currents in Xenopus oocytes expressing TRPM3α2, TRPM3γ2, or TRPM3γ3. Each column represents the mean ± SEM of 7–8 oocytes. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. *P < 0.05 versus TRPM3α2

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