Department of Otolaryngology - Head and Neck Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
St. Jude Children's Research Hospital, Department of Developmental Neurobiology, Memphis, TN, USA.
Sci Rep. 2019 May 3;9(1):6874. doi: 10.1038/s41598-019-43343-y.
Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin's C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin's exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17-18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17-18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.
耳蜗外毛细胞 (OHC) 表达运动蛋白 prestin，其对于敏感性和频率选择性是必需的。因为我们之前的工作表明，钙调蛋白结合位点 (CBS) 位于 prestin 的 C 端，特别是在无规卷曲区域内，我们试图删除 IDR 以研究钙依赖性、钙调蛋白结合对 OHC 功能的功能意义。尽管缺乏 IDR 的构建体 (∆IDR prestin) 在 HEK293T 细胞中表现出类似野生型的非线性电容 (NLC)，但 ∆IDR prestin 敲入 (KI) 的表型与 prestin 敲除相似：阈值升高，NLC 缺失，并且 OHC 从耳蜗的基底区域缺失。尽管测量了 ∆IDR prestin mRNA，但未检测到 prestin 蛋白。在 mRNA 水平上，prestin 的外显子 17 和 18 完全缺失，而不是编码 IDR 的较小区域。我们包含靶向缺失 (17-18 ∆IDR) 的杂交外显子未能在体外剪接，缺乏外显子 17 和 18 的 prestin 蛋白聚集并且未能靶向细胞膜。因此，∆IDR KI OHC 中 prestin 蛋白的缺失可能是由于杂交 17-18 ∆IDR 外显子的意外剪接，随后非功能性 prestin 蛋白迅速降解。
