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. 2015 Jul 21;5:12278. doi: 10.1038/srep12278

Figure 7. TRPV1 maximal activation by protons requires all four proton-binding subunits.

Figure 7

(a) Illustration of the different tetrameric concatemeric constructs used to study the stoichiometry for TRPV1 activation by protons. Black squares represent proton-binding intact subunits (wt) and grey squares represent subunits mutated in their proton-binding site in both positions E600Q and E648A (qa). (b) Whole-cell current traces (blue) in response to protons (pH 4; grey bar) of HEK293T cells transiently expressing the different mutant protons-binding concatemeric constructs, as indicated. Similar TRPV1 expression levels were estimated from the current evoked by saturating capsaicin concentration. Inset: representative whole-cell current traces (orange) in response to capsaicin (1 μM; light grey bar) of the 4wt and 4qa constructs. Note that the capsaicin response was unaffected by the mutation in the proton-binding sites. Holding potential of −40 mV. (c) Bar diagram representing the average ratio (±SEM) between protons (pH 4) - and capsaicin (at saturating concentration; 1 μM) - evoked amplitudes, normalized to the ratio obtained for the 4wt construct. Each bar represents 12–14 HEK293T cells transiently expressing the indicated concatemeric constructs. The statistical significance between the 4wt and the different proton-binding mutant constructs was determined with unpaired Student’s t test, where * represents P ≤ 0.05, **represents P ≤ 0.01 and ***represents P ≤ 0.001. Note a reduction in protons to capsaicin ratio with the inclusion of a single subunit mutated in its proton-binding sites (3wt/qa). (d) Normalized concentration-response relationships for protons of the 4wt and 3wt/qa concatemeric constructs. Each point represents the average (±SEM) response of 5–8 HEK293T cells transiently expressing the indicated concatemeric constructs. Solid lines are fit to the Hill equation (see Eq. (2)): 4wt (full circles, cyan line; nH = 1.8; EC50 = pH 5.23 ± 0.24), 3wt/qa (empty circle, blue line; nH = 1.6; EC50 = pH 5.20 ± 0.32). Holding potential of −40 mV.