Functional Studies of Deafness-Associated Pendrin and Prestin Variants

Abstract

Pendrin and prestin are evolutionary-conserved membrane proteins that are essential for normal hearing. Dysfunction of these proteins results in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here, we report results from our ongoing efforts to experimentally characterize pendrin and prestin variants using in vitro functional assays. With previously established fluorometric anion transport assays, we determined that many of the pendrin variants identified on transmembrane (TM) 10, which contains the essential anion binding site, and on the neighboring TM9 within the core domain resulted in impaired anion transport activity. We also determined the range of functional impairment in three deafness-associated prestin variants by measuring nonlinear capacitance (NLC), a proxy for motor function. Using the results from our functional analyses, we also evaluated the performance of AlphaMissense (AM), a computational tool for predicting the pathogenicity of missense variants. AM prediction scores correlated well with our experimental results; however, some variants were misclassified, underscoring the necessity of experimentally assessing the effects of variants. Together, our experimental efforts provide invaluable information regarding the pathogenicity of deafness-associated pendrin and prestin variants.

Keywords: DFNB4; DFNB61; SLC26A4; SLC26A5; hereditary hearing loss; nonlinear capacitance; pendred syndrome; pendrin; prestin.

Figure 3

Ala¹⁰⁰, Pro¹¹⁹, and Ser⁴⁴¹ sites in prestin. (A) 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 Ala¹⁰⁰, Pro¹¹⁹, and Ser⁴⁴¹ sites and bound chlorides are indicated by cyan, blue, purple, and red spheres, respectively. (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 Ala¹⁰⁰, Pro¹¹⁹, and Ser⁴⁴¹ in human prestin (hA5) and equivalents in others.

Figure 4

NLC measurements. (A) 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) Summaries of the NLC parameters (α, V_pk, and CD). Error bars indicate SD. ns, p ≥ 0.05; ** 0.001 < p ≤ 0.01; *** 0.0001 < p ≤ 0.001; **** p ≤ 0.0001.

Figure 5

The effects of missense changes at Ala¹⁰⁴ and Ala⁴⁵¹ on the anion transport function of pendrin. HCO₃⁻/Cl⁻ antiport assay conducted for p.A104V (left), p.A104T (left), p.A451G (right), p.A451S (right), and p.A451L (right) pendrin alongside WT control. Error bars indicate SD. Horizontal dotted lines indicate transport rates of uninduced cells. Solid lines indicate linear regressions (log₁₀ [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.

Figure 1

TM9-10 of pendrin. (A) 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) TM9 and TM10 region of the structure (residues 376–420), extracted from the right protomer in (A). 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. (C) 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 (A). The residues with asterisks (*) on top and gray shades indicate the locations of missense changes evaluated in Figure 2.

Figure 2

HCO₃⁻/Cl⁻ and I⁻/Cl⁻ antiport assays on pendrin variants. HCO₃⁻/Cl⁻ (A) and I⁻/Cl⁻ (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 (log₁₀ [Dox] vs. transport rates). Sample size information and statistics are provided in Table 1.

Figure 6

Correlation with AlphaMissense pathogenicity scores. HCO₃⁻/Cl⁻ transport rates of pendrin WT and variants from this study and the previous study [10] were normalized to the WT value and plotted against AlphaMissense (AM) pathogenicity scores [16]. Light blue shades indicate HCO₃⁻/Cl⁻ 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 HCO₃⁻/Cl⁻ antiport activity and the AM scores. Inset: Region indicated by the broken lines in left are enlarged to visualize variants with little or no HCO₃⁻/Cl⁻ transport activity.