Ectopic mouse TMC1 and TMC2 alone form mechanosensitive channels that are potently modulated by TMIE

• 成果类型: Article
• 署名作者: Chen, Yixuan; Li, Yulin; Liu, Yonghong; Sun, Jiawen; Feng, Wanying; Chen, Yanfei; Tian, Ye; Lei, Tianlun; Huang, Pingbo
• 署名单位: Hong Kong University of Science & Technology; Hong Kong University of Science & Technology; Hong Kong University of Science & Technology
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14320
• DOI: 10.1073/pnas.2403141122
• 发表日期: 2025-03-04
• 关键词: hair-cell; mechanotransduction-channel; ion selectivity; calcium-channel; transduction; site; pore; conductance; sensitivity; expression

摘要

The mechanotransduction (MT) channel expressed in cochlear and vestibular hair cells converts the mechanical stimulation of sound and head movements into electrochemical signals. Recently, TMC1 and TMC2 (TMC1/2) have been recognized as the pore- forming subunit of the MT channel, but TMC1/2 functional expression in heterologous cells-which is critical for unequivocally identifying them as the bona fide pore- forming subunit of the MT channel-has not been achieved because ectopic TMC1/2 become trapped in the ER. Here, we report that adding a Fyn lipidation tag to mouse TMC1/2 (mTMC1/2) drove their cell- surface expression, and, importantly, full- length mTMC1/2 expressed alone functioned as mechanosensitive channels, underscoring the view that TMC1/2 constitute the sole pore- forming subunit of the MT channel. Moreover, mouse transmembrane inner ear (TMIE) (mTMIE) protein robustly stimulated TMC1/2 channel activity by modulating their gating. Intriguingly, the N- terminal 27 residues of mTMIE were dispensable for regulating TMC1/2 in our in vitro functional assay, whereas, in striking contrast, mutating mTMIE C76C77, the predicted palmitoylation sites, eliminated mTMIE stimulation of mTMC1/2, indicating a crucial role of the palmitoyl group in regulating TMC1/2 gating. mTMC1/2+mTMIE form 18 pS and 24 pS single channels, respectively. mTMC1/2+mTMIE single channels showed biophysical and pharmacological properties similar to those of the MT channel. Our findings provide insights into several fundamental and debated aspects of the function of TMC1/2 and TMIE, and our functional assay of TMC1/2 and TMIE in heterologous cells will facilitate further functional and structural characterization of these proteins and other MT- complex components.