Functional Studies of Deafness-Associated Pendrin and Prestin Variants
<p>TM9-10 of pendrin. (<b>A</b>) The homodimeric structure of mouse pendrin (PDB: 7WK1). Protomers are shown in green and gray. Transmembrane and cytosolic domains are indicated in the lateral view (left). TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow. The bound chlorides are indicated by red spheres. In the extracellular view (right), the core domain of one of the protomers is shown in bright orange. (<b>B</b>) TM9 and TM10 region of the structure (residues 376–420), extracted from the right protomer in (<b>A</b>). TM9 (381–398) is shown in cyan, linker region (399–405) is in yellow, and TM10 (406–416) is in blue. Bound chloride is shown as a red sphere. (<b>C</b>) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5) showing the TM9 and TM10 region. Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends of the partial amino acid sequences. TM9 and TM10 are highlighted in cyan and blue, respectively, with connecting residues highlighted in yellow as in (<b>A</b>). The residues with asterisks (*) on top and gray shades indicate the locations of missense changes evaluated in <a href="#ijms-25-02759-f002" class="html-fig">Figure 2</a>.</p> "> Figure 2
<p>HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> and I<sup>−</sup>/Cl<sup>−</sup> antiport assays on pendrin variants. HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> (<b>A</b>) and I<sup>−</sup>/Cl<sup>−</sup> (<b>B</b>) antiport rates were plotted against doxycycline (Dox) concentration (0.1–10 µg/mL) for each mTq2-tagged pendrin variant alongside WT as indicated. Horizontal dotted lines indicate transport rates of uninduced cells. Error bars indicate SD. Solid lines indicate linear regressions (log<sub>10</sub> [Dox] vs. transport rates). Sample size information and statistics are provided in <a href="#ijms-25-02759-t001" class="html-table">Table 1</a>.</p> "> Figure 3
<p>Ala<sup>100</sup>, Pro<sup>119</sup>, and Ser<sup>441</sup> sites in prestin. (<b>A</b>) The homodimeric structure of human prestin (PDB: 7LGU). Protomers are shown in green and gray. In right panel, the core domain of one of the protomers is shown in bright orange. The Ala<sup>100</sup>, Pro<sup>119</sup>, and Ser<sup>441</sup> sites and bound chlorides are indicated by cyan, blue, purple, and red spheres, respectively. (<b>B</b>) Partial amino acid sequences of human and mouse pendrin (A4) and prestin (A5). Numbers in parentheses indicate the residue numbers at the N- and C-terminal ends. The residues with asterisks indicate Ala<sup>100</sup>, Pro<sup>119</sup>, and Ser<sup>441</sup> in human prestin (hA5) and equivalents in others.</p> "> Figure 4
<p>NLC measurements. (<b>A</b>) Examples of NLC recorded in HEK293T cells expressing WT, p.A100T, p.P119S, p.S441L, or p.S441A prestin. Different colors indicate individual recordings. (<b>B</b>) Summaries of the NLC parameters (α, V<sub>pk</sub>, and CD). Error bars indicate SD. ns, <span class="html-italic">p</span> ≥ 0.05; ** 0.001 < <span class="html-italic">p</span> ≤ 0.01; *** 0.0001 < <span class="html-italic">p</span> ≤ 0.001; **** <span class="html-italic">p</span> ≤ 0.0001.</p> "> Figure 5
<p>The effects of missense changes at Ala<sup>104</sup> and Ala<sup>451</sup> on the anion transport function of pendrin. HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> antiport assay conducted for p.A104V (<b>left</b>), p.A104T (<b>left</b>), p.A451G (<b>right</b>), p.A451S (<b>right</b>), and p.A451L (<b>right</b>) pendrin alongside WT control. Error bars indicate SD. Horizontal dotted lines indicate transport rates of uninduced cells. Solid lines indicate linear regressions (log<sub>10</sub> [Dox] vs. transport rates). Sample size information and statistics are provided in <a href="#ijms-25-02759-t001" class="html-table">Table 1</a>.</p> "> Figure 6
<p>Correlation with AlphaMissense pathogenicity scores. HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> transport rates of pendrin WT and variants from this study and the previous study [<a href="#B10-ijms-25-02759" class="html-bibr">10</a>] were normalized to the WT value and plotted against AlphaMissense (AM) pathogenicity scores [<a href="#B16-ijms-25-02759" class="html-bibr">16</a>]. Light blue shades indicate HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> rate of WT with errors (propagated errors). Gray shade between AM scores 0.34–0.56 marks the “ambiguous” category, with scores lower being “benign” and higher being “pathogenic” as indicated. Red line indicates the linear fit between the HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> antiport activity and the AM scores. Inset: Region indicated by the broken lines in left are enlarged to visualize variants with little or no HCO<sub>3</sub><sup>−</sup>/Cl<sup>−</sup> transport activity.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Functional Characterization of Pendrin Variants
2.2. Functional Characterization of Prestin Variants
Transport Activity [Mean ± SD (n)] | Dox-Dependence | ||||||||
---|---|---|---|---|---|---|---|---|---|
Dox: 0.1 µg/mL | Dox: 0.3 µg/mL | Dox: 1 µg/mL | Dox: 3 µg/mL | Dox: 10 µg/mL | Transport Activity/ log10 [Dox] (Slope ± SE) | Comparison to WT (F-Test) | |||
WT | HCO3–/Cl– | 0.597 ± 0.109 (10) | 0.795 ± 0.125 (10) | 1.102 ± 0.163 (10) | 1.485 ± 0.290 (10) | 1.731 ± 0.253 (10) | 0.591 ± 0.040 | not applicable | |
[nM/sec] | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | |||
I–/Cl– | 0.08 ± 0.04 (3) | 0.14 ± 0.01 (3) | 0.18 ± 0.02 (3) | 0.23 ± 0.04 (3) | 0.21 ± 0.02 (3) | 0.069 ± 0.012 | not applicable | ||
[mM/sec] | p = 0.049 | p = 0.0002 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | |||
Figure 2 | p.Gln383Glu (c.1147C>G) | HCO3–/Cl– | 0.061 ± 0.023 (4) | 0.072 ± 0.029 (4) | 0.084 ± 0.060 (4) | 0.125 ± 0.059 (3) | 0.073 ± 0.020 (4) | 0.015 ± 0.014 | Function impaired |
[nM/sec] | p = 0.4446 | p = 0.6253 | p = 0.8678 | p = 0.3807 | p = 0.6457 | p = 0.0034 | p < 0.0001 | ||
I–/Cl– | 0.01 ± 0.003 (3) | 0.012 ± 0.003 (3) | 0.011 ± 0.004 (3) | 0.015 ± 0.001 (3) | 0.017 ± 0.005 (3) | 0.003 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.2067 | p = 0.2589 | p = 0.2398 | p = 0.3427 | p = 0.4135 | p = 0.0132 | p < 0.0001 | ||
p.Glu384Gly (c.1151G>C) | HCO3–/Cl– | 0.055 ± 0.018 (4) | 0.063 ± 0.028 (4) | 0.081 ± 0.025 (4) | 0.088 ± 0.018 (4) | 0.081 ± 0.038 (4) | 0.016 ± 0.008 | Function impaired | |
[nM/sec] | p = 0.2875 | p = 0.407 | p = 0.7755 | p = 0.9276 | p = 0.7679 | p = 0.066 | p < 0.0001 | ||
I–/Cl– | 0.008 ± 0.002 (3) | 0.009 ± 0.002 (3) | 0.016 ± 0.009 (3) | 0.013 ± 0.004 (3) | 0.012 ± 0.002 (3) | 0.002 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.1742 | p = 0.1931 | p = 0.3773 | p = 0.3016 | p = 0.2592 | p = 0.1892 | p < 0.0001 | ||
p.Ala387Val (c.1160C>T) | HCO3–/Cl– | 0.046 ± 0.027 (4) | 0.054 ± 0.14 (4) | 0.067 ± 0.032 (4) | 0.082 ± 0.053 (4) | 0.100 ± 0.051 (4) | 0.028 ± 0.011 | Function impaired | |
[nM/sec] | p = 0.2327 | p = 0.3218 | p = 0.5252 | p = 0.8158 | p = 0.7995 | p = 0.023 | p < 0.0001 | ||
I–/Cl– | 0.008 ± 0.005 (3) | 0.016 ± 0.005 (3) | 0.011 ± 0.002 (3) | 0.017 ± 0.008 (3) | 0.015 ± 0.003 (3) | 0.003 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.1662 | p = 0.4038 | p = 0.2439 | p = 0.4111 | p = 0.3413 | p = 0.1819 | p < 0.0001 | ||
p.Gly389Arg (c.1165G>A) | HCO3–/Cl– | 0.054 ± 0.049 (3) | 0.064 ± 0.023 (3) | 0.064 ± 0.015 (3) | 0.071 ± 0.013 (3) | 0.075 ± 0.023 (3) | 0.010 ± 0.009 | Function impaired | |
[nM/sec] | p = 0.3825 | p = 0.5245 | p = 0.5265 | p = 0.6367 | p = 0.6956 | p = 0.3057 | p < 0.0001 | ||
I–/Cl– | 0.011 ± 0.007 (3) | 0.009 ± 0.005 (3) | 0.012 ± 0.006 (3) | 0.011 ± 0.001 (3) | 0.013 ± 0.004 (3) | 0.001 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.2336 | p = 0.1889 | p = 0.2642 | p = 0.2429 | p = 0.2878 | p = 0.4214 | p < 0.0001 | ||
p.Gly389Arg (c.1165G>C) | HCO3–/Cl– | 0.057 ± 0.015 (3) | 0.110 ± 0.090 (3) | 0.076 ± 0.019 (3) | 0.077 ± 0.012 (3) | 0.113 ± 0.066 (3) | 0.015 ± 0.018 | Function impaired | |
[nM/sec] | p = 0.4957 | p = 0.6993 | p = 0.7679 | p = 0.7755 | p = 0.6584 | p = 0.408 | p < 0.0001 | ||
I–/Cl– | 0.018 ± 0.008 (3) | 0.014 ± 0.009 (3) | 0.013 ± 0.002 (3) | 0.015 ± 0.002 (3) | 0.013 ± 0.002 (3) | slope < 0 | Function impaired | ||
[mM/sec] | p = 0.4607 | p = 0.3203 | p = 0.3036 | p = 0.375 | p = 0.2997 | p = 0.4403 | p < 0.0001 | ||
p.Ser391Asn (c.1172G>A) | HCO3–/Cl– | 0.076 ± 0.026 (4) | 0.084 ± 0.043 (4) | 0.075 ± 0.024 (4) | 0.085 ± 0.048 (4) | 0.066 ± 0.022 (4) | slope < 0 | Function impaired | |
[nM/sec] | p = 0.6844 | p = 0.8509 | p = 0.652 | p = 0.879 | p = 0.4908 | p = 0.6983 | p < 0.0001 | ||
I–/Cl– | 0.012 ± 0.004 (3) | 0.015 ± 0.005 (3) | 0.018 ± 0.001 (3) | 0.019 ± 0.007 (3) | 0.024 ± 0.006 (3) | 0.005 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.2746 | p = 0.3545 | p = 0.462 | p = 0.4873 | p = 0.7334 | p = 0.0053 | p < 0.0001 | ||
p.Ser391Arg (c.1173C>A) | HCO3–/Cl– | 0.049 ± 0.009 (3) | 0.038 ± 0.015 (3) | 0.036 ± 0.039 (3) | 0.056 ± 0.022 (3) | 0.106 ± 0.074 (3) | 0.026 ± 0.014 | Function impaired | |
[nM/sec] | p = 0.3588 | p = 0.2441 | p = 0.2289 | p = 0.4346 | p = 0.7391 | p = 0.0897 | p < 0.0001 | ||
I–/Cl– | 0.014 ± 0.002 (3) | 0.014 ± 0.003 (3) | 0.015 ± 0.001 (3) | 0.013 ± 0.002 (3) | 0.013 ± 0.001 (3) | slope < 0 | Function impaired | ||
[mM/sec] | p = 0.2964 | p = 0.3102 | p = 0.3437 | p = 0.2906 | p = 0.2652 | p = 0.4322 | p < 0.0001 | ||
p.Asn392Ser (c.1175A>G) | HCO3–/Cl– | 0.049 ± 0.018 (3) | 0.052 ± 0.046 (3) | 0.089 ± 0.041 (3) | 0.056 ± 0.032 (3) | 0.096 ± 0.027 (3) | 0.020 ± 0.012 | Function impaired | |
[nM/sec] | p = 0.3348 | p = 0.3763 | p = 0.9589 | p = 0.4347 | p = 0.9091 | p = 0.1275 | p < 0.0001 | ||
I–/Cl– | 0.009 ± 0.001 (3) | 0.016 ± 0.003 (3) | 0.015 ± 0.002 (3) | 0.015 ± 0.001 (3) | 0.023 ± 0.005 (3) | 0.005 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.1892 | p = 0.3664 | p = 0.3464 | p = 0.340 | p = 0.6698 | p = 0.0012 | p < 0.0001 | ||
p.Gly396Glu (c.1187G>A) | HCO3–/Cl– | 0.089 ± 0.038 (4) | 0.096 ± 0.060 (4) | 0.121 ± 0.054 (4) | 0.084 ± 0.031 (4) | 0.108 ± 0.013 (4) | 0.006 ± 0.013 | Function impaired | |
[nM/sec] | p = 0.9512 | p = 0.9048 | p = 0.4323 | p = 0.852 | p = 0.6605 | p = 0.6755 | p < 0.0001 | ||
I–/Cl– | 0.009 ± 0.005 (3) | 0.009 ± 0.001 (3) | 0.012 ± 0.002 (3) | 0.011 ± 0.003 (3) | 0.010 ± 0.002 (3) | 0.001 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.1751 | p = 0.1868 | p = 0.2536 | p = 0.2304 | p = 0.2133 | p = 0.3095 | p < 0.0001 | ||
p.Val402Met (c.1204G>A) | HCO3–/Cl– | 0.086 ± 0.026 (3) | 0.075 ± 0.010 (3) | 0.072 ± 0.029 (3) | 0.108 ± 0.071 (3) | 0.095 ± 0.020 (3) | 0.010 ± 0.013 | Function impaired | |
[nM/sec] | p = 0.9029 | p = 0.7134 | p = 0.6663 | p = 0.6978 | p = 0.9317 | p = 0.4368 | p < 0.0001 | ||
I–/Cl– | 0.013 ± 0.001 (3) | 0.011 ± 0.003 (3) | 0.016 ± 0.007 (3) | 0.018 ± 0.004 (3) | 0.018 ± 0.003 (3) | 0.004 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.2741 | p = 0.2302 | p = 0.3718 | p = 0.4644 | p = 0.4446 | p = 0.0321 | p < 0.0001 | ||
p.Thr404Ile (c.1211C>T) | HCO3–/Cl– | 0.091 ± 0.032 (5) | 0.062 ± 0.059 (5) | 0.085 ± 0.043 (5) | 0.094 ± 0.059 (5) | 0.100 ± 0.047 (5) | 0.010 ± 0.013 | Function impaired | |
[nM/sec] | p = 0.9989 | p = 0.4374 | p = 0.870 | p = 0.940 | p = 0.8092 | p = 0.4618 | p < 0.0001 | ||
I–/Cl– | 0.011 ± 0.004 (3) | 0.011 ± 0.004 (3) | 0.014 ± 0.004 (3) | 0.015 ± 0.003 (3) | 0.012 ± 0.007 (3) | 0.001 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.2324 | p = 0.2375 | p = 0.3348 | p = 0.365 | p = 0.2664 | p = 0.4046 | p < 0.0001 | ||
p.Ala406Thr (c.1216G>A) | HCO3–/Cl– | 0.233 ± 0.068 (3) | 0.345 ± 0.073 (3) | 0.473 ± 0.053 (3) | 0.479 ± 0.026 (3) | 0.461 ± 0.088 (3) | 0.118 ± 0.028 | Function impaired | |
[nM/sec] | p = 0.0172 | p = 0.0002 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0011 | p < 0.0001 | ||
I–/Cl– | 0.056 ± 0.009 (3) | 0.094 ± 0.021 (3) | 0.120 ± 0.037 (3) | 0.111 ± 0.040 (3) | 0.122 ± 0.033 (3) | 0.030 ± 0.011 | Function impaired | ||
[mM/sec] | p = 0.3394 | p = 0.0308 | p = 0.005 | p = 0.0095 | p = 0.0046 | p = 0.017 | p = 0.0212 | ||
p.Ser408Asp (c.1222_3TC>GA) * | HCO3–/Cl– | 0.040 ± 0.019 (4) | 0.057 ± 0.020 (4) | 0.053 ± 0.012 (4) | 0.070 ± 0.031 (4) | 0.088 ± 0.031 (4) | 0.022 ± 0.007 | Function impaired | |
[nM/sec] | p = 0.1339 | p = 0.3078 | p = 0.2554 | p = 0.5357 | p = 0.919 | p = 0.0068 | p < 0.0001 | ||
I–/Cl– | 0.011 ± 0.002 (3) | 0.013 ± 0.003 (3) | 0.017 ± 0.004 (3) | 0.016 ± 0.007 (3) | 0.019 ± 0.002 (3) | 0.004 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.2394 | p = 0.276 | p = 0.4036 | p = 0.3921 | p = 0.4893 | p = 0.0165 | p < 0.0001 | ||
p.Ser408Glu (c.1222_4TCC>GAG) * | HCO3–/Cl– | 0.066 ± 0.032 (5) | 0.075 ± 0.021 (5) | 0.063 ± 0.034 (5) | 0.084 ± 0.059 (5) | 0.091 ± 0.057 (5) | 0.012 ± 0.012 | Function impaired | |
[nM/sec] | p = 0.477 | p = 0.6504 | p = 0.427 | p = 0.8493 | p = 0.9929 | p = 0.3294 | p < 0.0001 | ||
I–/Cl– | 0.008 ± 0.003 (3) | 0.009 ± 0.005 (3) | 0.010 ± 0.002 (3) | 0.015 ± 0.001 (3) | 0.010 ± 0.001 (3) | 0.002 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.1561 | p = 0.1915 | p = 0.2025 | p = 0.3308 | p = 0.2164 | p = 0.0695 | p < 0.0001 | ||
p.Arg409Cys (c.1225C>T) | HCO3–/Cl– | 0.103 ± 0.066 (3) | 0.091 ± 0.081 (3) | 0.065 ± 0.036 (3) | 0.076 ± 0.040 (3) | 0.168 ± 0.085 (3) | 0.023 ± 0.024 | Function impaired | |
[nM/sec] | p = 0.8259 | p = 0.9943 | p = 0.6379 | p = 0.782 | p = 0.1685 | p = 0.3529 | p < 0.0001 | ||
I–/Cl– | 0.022 ± 0.002 (3) | 0.024 ± 0.002 (3) | 0.028 ± 0.003 (3) | 0.028 ± 0.004 (3) | 0.026 ± 0.004 (3) | 0.002 ± 0.001 | Function impaired | ||
[mM/sec] | p = 0.6456 | p = 0.7489 | p = 0.9292 | p = 0.9227 | p = 0.8445 | p = 0.0791 | p < 0.0001 | ||
p.Arg409Leu (c.1226G>T) | HCO3–/Cl– | 0.234 ± 0.042 (4) | 0.275 ± 0.037 (4) | 0.318 ± 0.043 (4) | 0.334 ± 0.082 (4) | 0.380 ± 0.044 (3) | 0.071 ± 0.016 | Function impaired | |
[nM/sec] | p = 0.225 | p = 0.1221 | p = 0.0603 | p = 0.0453 | p = 0.0003 | p = 0.0004 | p < 0.0001 | ||
I–/Cl– | 0.061 ± 0.002 (3) | 0.078 ± 0.005 (3) | 0.086 ± 0.003 (3) | 0.086 ± 0.005 (3) | 0.095 ± 0.013 (3) | 0.015 ± 0.003 | Function impaired | ||
[mM/sec] | p = 0.0552 | p = 0.0072 | p = 0.0027 | p = 0.0024 | p = 0.009 | p < 0.0001 | p = 0.0001 | ||
p.Ala411Pro (c.1231G>C) | HCO3–/Cl– | 0.064 ± 0.021 (3) | 0.084 ± 0.021 (3) | 0.071 ± 0.033 (3) | 0.066 ± 0.013 (3) | 0.065 ± 0.014 (3) | slope < 0 | Function impaired | |
[nM/sec] | p = 0.5137 | p = 0.8711 | p = 0.6193 | p = 0.5427 | p = 0.529 | p = 0.6639 | p < 0.0001 | ||
I–/Cl– | 0.016 ± 0.002 (3) | 0.024 ± 0.002 (3) | 0.027 ± 0.005 (3) | 0.028 ± 0.005 (3) | 0.029 ± 0.005 (3) | 0.006 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.3929 | p = 0.7402 | p = 0.8987 | p = 0.9163 | p = 0.9784 | p = 0.0033 | p < 0.0001 | ||
p.Ala411Thr (c.1231G>A) | HCO3–/Cl– | 0.890 ± 0.010 (3) | 1.176 ± 0.035 (3) | 1.370 ± 0.120 (3) | 1.732 ± 0.237 (3) | 1.752 ± 0.047 (3) | 0.455 ± 0.049 | WT-like | |
[nM/sec] | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0847 | ||
I–/Cl– | 0.064 ± 0.012 (3) | 0.079 ± 0.014 (3) | 0.094 ± 0.011 (3) | 0.113 ± 0.023 (3) | 0.123 ± 0.022 (3) | 0.030 ± 0.006 | Function impaired | ||
[mM/sec] | p = 0.0955 | p = 0.0224 | p = 0.0054 | p = 0.0009 | p = 0.0004 | p = 0.0001 | p = 0.0062 | ||
p.Val412Ile (c.1234G>A) | HCO3–/Cl– | 0.868 ± 0.186 (3) | 1.132 ± 0.503 (3) | 1.430 ± 0.706 (3) | 1.530 ± 0.532 (3) | 2.081 ± 0.849 (3) | 0.566 ± 0.194 | WT-like | |
[nM/sec] | p = 0.041 | p = 0.0091 | p = 0.0016 | p = 0.0009 | p < 0.0001 | p = 0.0121 | p = 0.8432 | ||
I–/Cl– | 0.085 ± 0.009 (3) | 0.117 ± 0.005 (3) | 0.122 ± 0.016 (3) | 0.141 ± 0.015 (3) | 0.146 ± 0.006 (3) | 0.029 ± 0.004 | Function impaired | ||
[mM/sec] | p = 0.005 | p = 0.0002 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0036 | ||
p.Gln413Pro (c.1238A>C) | HCO3–/Cl– | 0.095 ± 0.021 (3) | 0.081 ± 0.027 (3) | 0.090 ± 0.057 (3) | 0.144 ± 0.056 (3) | 0.121 ± 0.100 (3) | 0.023 ± 0.020 | Function impaired | |
[nM/sec] | p = 0.9349 | p = 0.8519 | p = 0.984 | p = 0.3123 | p = 0.5596 | p = 0.2745 | p < 0.0001 | ||
I–/Cl– | 0.019 ± 0.003 (3) | 0.026 ± 0.007 (3) | 0.027 ± 0.002 (3) | 0.024 ± 0.001 (3) | 0.022 ± 0.004 (3) | 0.001 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.4857 | p = 0.8454 | p = 0.8783 | p = 0.7352 | p = 0.6318 | p = 0.6167 | p < 0.0001 | ||
p.Gln413Arg (c.1238A>G) | HCO3–/Cl– | 0.313 ± 0.107 (3) | 0.349 ± 0.156 (3) | 0.440 ± 0.212 (3) | 0.459 ± 0.203 (3) | 0.424 ± 0.208 (3) | 0.066 ± 0.059 | Function impaired | |
[nM/sec] | p = 0.0652 | p = 0.0349 | p = 0.0069 | p = 0.0048 | p = 0.0092 | p = 0.2848 | p < 0.0001 | ||
I–/Cl– | 0.039 ± 0.011 (3) | 0.050 ± 0.009 (3) | 0.058 ± 0.002 (3) | 0.064 ± 0.002 (3) | 0.072 ± 0.007 (3) | 0.016 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.5267 | p = 0.1857 | p = 0.0756 | p = 0.0391 | p = 0.0139 | p < 0.0001 | p = 0.0001 | ||
p.Glu414Lys (c.1240G>A) | HCO3–/Cl– | 0.305 ± 0.075 (3) | 0.314 ± 0.166 (3) | 0.327 ± 0.079 (3) | 0.404 ± 0.106 (3) | 0.429 ± 0.262 (3) | 0.067 ± 0.050 | Function impaired | |
[nM/sec] | p = 0.0431 | p = 0.0356 | p = 0.0279 | p = 0.0055 | p = 0.0033 | p = 0.1998 | p < 0.0001 | ||
I–/Cl– | 0.040 ± 0.008 (3) | 0.045 ± 0.007 (3) | 0.049 ± 0.002 (3) | 0.051 ± 0.005 (3) | 0.061 ± 0.008 (3) | 0.010 ± 0.002 | Function impaired | ||
[mM/sec] | p = 0.4681 | p = 0.3109 | p = 0.2121 | p = 0.1617 | p = 0.0545 | p = 0.0007 | p < 0.0001 | ||
p.Ser415Gly (c.1243A>G) | HCO3–/Cl– | 0.384 ± 0.171 (3) | 0.484 ± 0.075 (3) | 0.727 ± 0.184 (3) | 0.824 ± 0.260 (3) | 0.774 ± 0.260 (3) | 0.224 ± 0.070 | Function impaired | |
[nM/sec] | p = 0.0303 | p = 0.0059 | p = 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0072 | p < 0.0001 | ||
I–/Cl– | 0.068 ± 0.011 (3) | 0.089 ± 0.011 (3) | 0.097 ± 0.014 (3) | 0.107 ± 0.016 (3) | 0.112 ± 0.007 (3) | 0.021 ± 0.004 | Function impaired | ||
[mM/sec] | p = 0.0377 | p = 0.0039 | p = 0.0017 | p = 0.0006 | p = 0.0003 | p = 0.0002 | p = 0.0007 | ||
p.Ser415Arg (c.1245C>A) | HCO3–/Cl– | 0.634 ± 0.135 (3) | 0.923 ± 0.072 (3) | 0.915 ± 0.013 (3) | 1.084 ± 0.041 (3) | 1.242 ± 0.057 (3) | 0.275 ± 0.033 | Function impaired | |
[nM/sec] | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | ||
I–/Cl– | 0.052 ± 0.009 (3) | 0.069 ± 0.018 (3) | 0.080 ± 0.011 (3) | 0.094 ± 0.010 (3) | 0.091 ± 0.013 (3) | 0.021 ± 0.004 | Function impaired | ||
[mM/sec] | p = 0.203 | p = 0.0371 | p = 0.0102 | p = 0.0023 | p = 0.0033 | p = 0.0005 | p = 0.0006 | ||
p.Ser532Ile (c.1595G>T) | HCO3–/Cl– | 0.549 ± 0.091 (4) | 0.793 ± 0.254 (4) | 0.816 ± 0.107 (4) | 0.845 ± 0.267 (4) | 0.912 ± 0.245 (4) | 0.155 ± 0.063 | Function impaired | |
[nM/sec] | p = 0.0011 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0244 | p < 0.0001 | ||
I–/Cl– | 0.053 ± 0.011 (3) | 0.055 ± 0.011 (3) | 0.069 ± 0.018 (3) | 0.086 ± 0.028 (3) | 0.095 ± 0.009 (3) | 0.023 ± 0.006 | Function impaired | ||
[mM/sec] | p = 0.2373 | p = 0.198 | p = 0.0572 | p = 0.0108 | p = 0.0045 | p = 0.0011 | p = 0.0015 | ||
Figure 5 | p.Ala104Val (c.311C>T) | HCO3–/Cl– | 0.079 ± 0.017 (4) | 0.152 ± 0.080 (4) | 0.110 ± 0.035 (4) | 0.132 ± 0.029 (4) | 0.105 ± 0.039 (4) | 0.006 ± 0.015 | Function impaired |
[nM/sec] | p = 0.7598 | p = 0.1325 | p = 0.6295 | p = 0.3075 | p = 0.718 | p = 0.7015 | p < 0.0001 | ||
p.Ala104Thr (c.310G>A) * | HCO3–/Cl– | 0.363 ± 0.051 (6) | 0.402 ± 0.075 (6) | 0.468 ± 0.039 (6) | 0.528 ± 0.100 (6) | 0.443 ± 0.084 (6) | 0.057 ± 0.022 | Function impaired | |
[nM/sec] | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.0159 | p < 0.0001 | ||
p.Ala451Gly (c.1352C>G) * | HCO3–/Cl– | 0.479 ± 0.092 (3) | 0.911 ± 0.063 (3) | 0.993 ± 0.129 (3) | 1.284 ± 0.089 (3) | 1.836 ± 0.184 (3) | 0.618 ± 0.056 | WT-like | |
[nM/sec] | p = 0.0062 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.7423 | ||
p.Ala451Ser (c.1351G>T) * | HCO3–/Cl– | 0.572 ± 0.140 (3) | 0.921 ± 0.162 (3) | 1.168 ± 0.127 (3) | 1.406 ± 0.167 (3) | 1.749 ± 0.251 (3) | 0.568 ± 0.057 | WT-like | |
[nM/sec] | p = 0.0027 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p < 0.0001 | p = 0.7667 | ||
p.Ala451Leu (c.1351_2GC>CT) * | HCO3–/Cl– | 0.097 ± 0.047 (3) | 0.144 ± 0.013 (3) | 0.100 ± 0.036 (3) | 0.093 ± 0.014 (3) | 0.143 ± 0.064 (3) | 0.008 ± 0.015 | Function impaired | |
[nM/sec] | p = 0.3455 | p = 0.0283 | p = 0.3058 | p = 0.4034 | p = 0.0299 | p = 0.6021 | p < 0.0001 |
2.3. Comparison to AlphaMissense Variant Effect Predictor
3. Discussion
4. Materials and Methods
4.1. Generation of Stable Cell Lines
4.2. Fluorometric Anion Transport Assays
4.3. Whole-Cell Recordings
4.4. NLC Data Analysis
4.5. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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HCO3–/Cl– Antiport Assay Results | AM Prediction | |||||
---|---|---|---|---|---|---|
%WT Activity | Variant Effect | AM Score | AM Category | |||
Wasano et al., 2020 [10] | p.Ser28Gly (c.82A>G) | 29.2 ± 8.3 | Function impaired | 0.1421 | benign | |
p.Ser49Arg (c.147C>G) | 104.1 ± 13.7 | WT-like | 0.0831 | benign | ||
p.Pro76Ser (c.226C>T) | 30.5 ± 6.3 | Function impaired | 0.2937 | benign | ||
p.Ser90Leu (c.269C>T) | 0.1 ± 0.9 | Function impaired | 0.8985 | pathogenic | ||
p.Thr99Arg (c.296C>G) | 0 | Function impaired | 0.9802 | pathogenic | ||
p.Leu117Phe (c.349C>T) | 95.5 ± 12.5 | WT-like | 0.804 | pathogenic | ||
p.Pro123Ser (c.367C>T) | 2.9 ± 1.3 | Function impaired | 0.7286 | pathogenic | ||
p.Gly131Val (c.392G>T) | 0.4 ± 1.7 | Function impaired | 0.9925 | pathogenic | ||
p.Ser133Thr (c.397T>A) | 2.3 ± 2.0 | Function impaired | 0.4642 | ambiguous | ||
p.Gly139Ala (c.416G>C) | 0 | Function impaired | 0.9105 | pathogenic | ||
p.Met147Thr (c.440T>C) | 0.6 ± 2.8 | Function impaired | 0.9459 | pathogenic | ||
p.Met147Val (c.439A>G) | 6.4 ± 8.5 | Function impaired | 0.7909 | pathogenic | ||
p.Val163Ile (c.487G>A) | 105.1 ± 23.2 | WT-like | 0.0632 | benign | ||
p.Val186Phe (c.556G>T) | 0 | Function impaired | 0.4777 | ambiguous | ||
p.Thr193Ile (c.578C>T) | 0.9 ± 1.3 | Function impaired | 0.9082 | pathogenic | ||
p.Tyr214Cys (c.641A>G) | 31.4 ± 6.0 | Function impaired | 0.8371 | pathogenic | ||
p.Val239Asp (c.716T>A) | 4.9 ± 2.3 | Function impaired | 0.9612 | pathogenic | ||
p.Asp266Asn (c.796G>A) | 106.7 ± 17.7 | WT-like | 0.0528 | ambiguous | ||
p.Thr307Ala (c.919A>G) | 105.5 ± 15.2 | WT-like | 0.4074 | ambiguous | ||
p.Asn324Tyr (c.970A>T) | 99.4 ± 19.5 | WT-like | 0.1773 | pathogenic | ||
p.Gly334Val (c.1001G>T) | 59.5 ± 10.8 | Function impaired | 0.7329 | pathogenic | ||
p.Phe354Ser (c.1061T>C) | 96.6 ± 17.4 | WT-like | 0.5739 | pathogenic | ||
p.Lys369Glu (c.1105A>G) | 39.4 ± 4.2 | Function impaired | 0.4968 | ambiguous | ||
p.Ala372Val (c.1115C>T) | 0.1± 0.9 | Function impaired | 0.9528 | pathogenic | ||
p.Asn392Tyr (c.1174A>T) | 0.5 ± 0.7 | Function impaired | 0.9493 | pathogenic | ||
p.Ser399Pro (c.1195T>C) | 78.9 ± 12.2 | Function impaired | 0.5658 | pathogenic | ||
p.Ser408Phe (c.1223C>T) | 0.9 ± 1.3 | Function impaired | 0.9938 | pathogenic | ||
p.Arg409His (c.1226G>A) | 2.8 ± 2.6 | Function impaired | 0.9499 | pathogenic | ||
p.Thr410Lys (c.1229C>A) | 1.9 ± 1.1 | Function impaired | 0.9957 | pathogenic | ||
p.Thr410Met (c.1229C>T) | 1.4 ± 1.7 | Function impaired | 0.9314 | pathogenic | ||
p.Thr416Pro (c.1246A>C) | 2.2 ± 0.7 | Function impaired | 0.8985 | pathogenic | ||
p.Gln421Leu (c.1262A>T) | 14.1 ± 5.9 | Function impaired | 0.8205 | pathogenic | ||
p.Ile426Asn (c.1277T>A) | 63.8 ± 11.2 | Function impaired | 0.8501 | pathogenic | ||
p.Leu445Trp (c.1334T>G) | 0 | Function impaired | 0.9933 | pathogenic | ||
p.Asn457Lys (c.1371C>A) | 36.4 ± 5.5 | Function impaired | 0.992 | pathogenic | ||
p.Arg470His (c.1409G>A) | 119.6 ± 19.7 | WT-like | 0.0709 | benign | ||
p.Val483Glu (c.1448T>A) | 70.7 ± 11.6 | WT-like | 0.7757 | pathogenic | ||
p.Gly497Ser (c.1489G>A) | 9.8 ± 2.9 | Function impaired | 0.883 | pathogenic | ||
p.Thr527Pro (c.1579A>C) | 42.0 ± 6.8 | Function impaired | 0.7253 | pathogenic | ||
p.Ile529Ser (c.1586T>G) | 16.1 ± 3.1 | Function impaired | 0.7726 | pathogenic | ||
p.Tyr556Cys (c.1667A>G) | 6.5 ± 1.3 | Function impaired | 0.4069 | ambiguous | ||
p.Cys565Tyr (c.1694G>A) | 80.8 ± 10.3 | WT-like | 0.1986 | benign | ||
p.Ser657Asn (c.1970G>A) | 33.0 ± 6.0 | Function impaired | 0.7648 | pathogenic | ||
p.Val659Leu (c.1975G>C) | 37.9 ± 8.1 | Function impaired | 0.2582 | benign | ||
p.Ser666Phe (c.1997C>T) | 17.8 ± 5.3 | Function impaired | 0.4588 | ambiguous | ||
p.Asp669Glu (c.2007C>A) | 0.0 ± 0.0 | Function impaired | 0.973 | pathogenic | ||
p.Phe683Ser (c.2048T>C) | 2.8 ± 1.8 | Function impaired | 0.9461 | pathogenic | ||
p.Phe692Leu (c.2074T>C) | 36.0 ± 9.6 | Function impaired | 0.7169 | pathogenic | ||
p.Leu703Pro (c.2108T>C) | 0.3 ± 0.9 | Function impaired | 0.955 | pathogenic | ||
p.Thr721Met (c.2162C>T) | 0.6 ± 1.7 | Function impaired | 0.4373 | ambiguous | ||
p.His723Arg (c.2168A>G) | 13.4 ± 6.2 | Function impaired | 0.684 | pathogenic | ||
This study | p.Gln383Glu (c.1147C>G) | 2.5 ± 2.4 | Function impaired | 0.4658 | ambiguous | |
p.Glu384Gly (c.1151G>C) | 2.7 ± 1.4 | Function impaired | 0.9808 | pathogenic | ||
p.Ala387Val (c.1160C>T) | 4.7 ± 1.9 | Function impaired | 0.9179 | pathogenic | ||
p.Gly389Arg (c.1165G>A) | 1.7 ± 1.5 | Function impaired | 0.996 | pathogenic | ||
p.Gly389Arg (c.1165G>C) | 2.5 ± 3.1 | Function impaired | 0.996 | pathogenic | ||
p.Ser391Asn (c.1172G>A) | 0 | Function impaired | 0.8762 | pathogenic | ||
p.Ser391Arg (c.1173C>A) | 4.3 ± 2.4 | Function impaired | 0.9974 | pathogenic | ||
p.Asn392Ser (c.1175A>G) | 3.4 ± 2.0 | Function impaired | 0.3199 | benign | ||
p.Gly396Glu (c.1187G>A) | 1.0 ± 0.2 | Function impaired | 0.9866 | pathogenic | ||
p.Val402Met (c.1204G>A) | 1.7 ± 2.2 | Function impaired | 0.5617 | pathogenic | ||
p.Thr404Ile (c.1211C>T) | 1.7 ± 2.2 | Function impaired | 0.9734 | pathogenic | ||
p.Ala406Thr (c.1216G>A) | 20.0 ± 4.9 | Function impaired | 0.8845 | pathogenic | ||
p.Ser408Asp (c. 1222_3TC>GA) * | 3.7 ± 1.2 | Function impaired | 0.9971 | pathogenic | ||
p.Ser408Glu (c.1222_4TCC>GAG) * | 2.0 ± 2.0 | Function impaired | 0.9964 | pathogenic | ||
p.Arg409Cys (c.1225C>T) | 3.4 ± 4.1 | Function impaired | 0.8893 | pathogenic | ||
p.Arg409Leu (c.1226G>T) | 12.0 ± 2.8 | Function impaired | 0.9763 | pathogenic | ||
p.Ala411Pro (c.1231G>C) | 0 | Function impaired | 0.9893 | pathogenic | ||
p.Ala411Thr (c.1231G>A) | 77.0 ± 9.8 | WT-like | 0.5393 | ambiguous | ||
p.Val412Ile (c.1234G>A) | 95.8 ± 33.5 | WT-like | 0.0792 | benign | ||
p.Gln413Pro (c.1238A>C) | 3.9 ± 3.4 | Function impaired | 0.9839 | pathogenic | ||
p.Gln413Arg (c.1238A>G) | 11.2 ± 10.0 | Function impaired | 0.8859 | pathogenic | ||
p.Glu414Lys (c.1240G>A) | 11.3 ± 8.5 | Function impaired | 0.8309 | pathogenic | ||
p.Ser415Gly (c.1243A>G) | 37.9 ± 12.1 | Function impaired | 0.1027 | benign | ||
p.Ser415Arg (c.1245C>A) | 46.5 ± 6.4 | Function impaired | 0.9514 | pathogenic | ||
p.Ser532Ile (c.1595G>T) | 26.2 ± 10.8 | Function impaired | 0.3304 | benign | ||
p.Ala104Val (c.311C>T) | 1.0 ± 2.5 | Function impaired | 0.9684 | pathogenic | ||
p.Ala104Thr (c.310G>A) * | 9.6 ± 3.8 | Function impaired | 0.9153 | pathogenic | ||
p.Ala451Gly (c.1352C>G) * | 104.6 ± 11.8 | WT-like | 0.1774 | benign | ||
p.Ala451Ser (c.1351G>T) * | 96.1 ± 11.6 | WT-like | 0.2429 | benign | ||
p.Ala451Leu (c.1351_2GC>CT) * | 1.4 ± 2.5 | Function impaired | 0.9707 | pathogenic |
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Takahashi, S.; Kojima, T.; Wasano, K.; Homma, K. Functional Studies of Deafness-Associated Pendrin and Prestin Variants. Int. J. Mol. Sci. 2024, 25, 2759. https://doi.org/10.3390/ijms25052759
Takahashi S, Kojima T, Wasano K, Homma K. Functional Studies of Deafness-Associated Pendrin and Prestin Variants. International Journal of Molecular Sciences. 2024; 25(5):2759. https://doi.org/10.3390/ijms25052759
Chicago/Turabian StyleTakahashi, Satoe, Takashi Kojima, Koichiro Wasano, and Kazuaki Homma. 2024. "Functional Studies of Deafness-Associated Pendrin and Prestin Variants" International Journal of Molecular Sciences 25, no. 5: 2759. https://doi.org/10.3390/ijms25052759