STOM binding at the distal C-terminus of ASIC3 is necessary for inhibition.(A–F) Each column shows the cartoon of the chimera being tested. In the case of D–F, the cartoon shows a blow up of the distal C-terminus. In addition, each column has a representative set of pH 5.5–evoked currents of the indicated chimeric channel alone (black) and cotransfected with STOM (red). Finally, each column shows a boxplot of the current density of the channel with and without STOM coexpression. (A) Average current densities for ASIC3(466–533)1a in the absence (black) and presence (red) of STOM were 99.5 ± 26.2 pA/pF (n = 22) and 121.4 ± 41.7 pA/pF (n = 9), respectively. (B) Average current densities for ASIC3(466–505)1a in the absence (black) and presence (red) of STOM were 239.9 ± 80.3 pA/pF (n = 9) and 13.4 ± 5.1 pA/pF (n = 9), respectively. (C) Average current densities for ASIC3(506–533)1a in the absence (black) and presence (red) of STOM were 69.6 ± 18.7 pA/pF (n = 19) and 193.1 ± 61.8 pA/pF (n = 18), respectively. (D) Average current densities of ASIC3(506–519)1a-CER in the absence (black) and presence (red) of STOM-YFP were 42.6 ± 6.3 pA/pF (n = 23) and 12.0 ± 2.7 pA/pF (n = 19), respectively. (E) Average current densities of ASIC3(520–533)1a-CER in the absence (black) and presence (red) of STOM-YFP were 750.1 ± 118.7 pA/pF (n = 10) and 661.9 ± 101.2 pA/pF (n = 9), respectively. (F) Average current densities of ASIC3(526–533)1a-CER in the absence (black) and presence (red) of STOM-YFP were 586.3 ± 152.7 pA/pF (n = 6) STOM-YFP mean 572.9 ± 64.2 pA/pF (n = 7), respectively. (G) Plot showing the FRET efficiency for each chimeric construct. Average FRET efficiency for ASIC3(466–533)1a-CER + STOM-YFP = 1.0 ± 0.5% (n = 8). Average FRET efficiency for ASIC3(466–505)1a-CER and STOM-YFP = 13.1 ± 1.0% (n = 7). Average FRET efficiency for ASIC3(506–533)-CER + STOM-YFP = 1.5 ± 0.5% (n = 7). Average FRET efficiency for ASIC3(506–519)1a-CER + STOM-YFP = 9.9 ± 1.1% (n = 12). Average FRET efficiency for ASIC3(520–533)1a-CER + STOM-YFP = 1.1 ± 0.5% (n = 5). Average FRET efficiency for ASIC3(526–533)1a-CER + STOM-YFP = 0.2 ± 0.3% (n = 7). Dotted line in FRET plot corresponds to control WT ASIC3-CER + STOM-YFP FRET signal replotted from Fig. 1 E for comparison. Data are given as mean ± SEM.
Figure 3.

STOM binding at the distal C-terminus of ASIC3 is necessary for inhibition. (A – F) Each column shows the cartoon of the chimera being tested. In the case of D–F, the cartoon shows a blow up of the distal C-terminus. In addition, each column has a representative set of pH 5.5–evoked currents of the indicated chimeric channel alone (black) and cotransfected with STOM (red). Finally, each column shows a boxplot of the current density of the channel with and without STOM coexpression. (A) Average current densities for ASIC3(466–533)1a in the absence (black) and presence (red) of STOM were 99.5 ± 26.2 pA/pF (n = 22) and 121.4 ± 41.7 pA/pF (n = 9), respectively. (B) Average current densities for ASIC3(466–505)1a in the absence (black) and presence (red) of STOM were 239.9 ± 80.3 pA/pF (n = 9) and 13.4 ± 5.1 pA/pF (n = 9), respectively. (C) Average current densities for ASIC3(506–533)1a in the absence (black) and presence (red) of STOM were 69.6 ± 18.7 pA/pF (n = 19) and 193.1 ± 61.8 pA/pF (n = 18), respectively. (D) Average current densities of ASIC3(506–519)1a-CER in the absence (black) and presence (red) of STOM-YFP were 42.6 ± 6.3 pA/pF (n = 23) and 12.0 ± 2.7 pA/pF (n = 19), respectively. (E) Average current densities of ASIC3(520–533)1a-CER in the absence (black) and presence (red) of STOM-YFP were 750.1 ± 118.7 pA/pF (n = 10) and 661.9 ± 101.2 pA/pF (n = 9), respectively. (F) Average current densities of ASIC3(526–533)1a-CER in the absence (black) and presence (red) of STOM-YFP were 586.3 ± 152.7 pA/pF (n = 6) STOM-YFP mean 572.9 ± 64.2 pA/pF (n = 7), respectively. (G) Plot showing the FRET efficiency for each chimeric construct. Average FRET efficiency for ASIC3(466–533)1a-CER + STOM-YFP = 1.0 ± 0.5% (n = 8). Average FRET efficiency for ASIC3(466–505)1a-CER and STOM-YFP = 13.1 ± 1.0% (n = 7). Average FRET efficiency for ASIC3(506–533)-CER + STOM-YFP = 1.5 ± 0.5% (n = 7). Average FRET efficiency for ASIC3(506–519)1a-CER + STOM-YFP = 9.9 ± 1.1% (n = 12). Average FRET efficiency for ASIC3(520–533)1a-CER + STOM-YFP = 1.1 ± 0.5% (n = 5). Average FRET efficiency for ASIC3(526–533)1a-CER + STOM-YFP = 0.2 ± 0.3% (n = 7). Dotted line in FRET plot corresponds to control WT ASIC3-CER + STOM-YFP FRET signal replotted from Fig. 1 E for comparison. Data are given as mean ± SEM.

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