Department of Chemical Sciences, Indian Institute of Science Education and Research Mohali, Mohali, Punjab 140306, India.
Institute of Nano Science and Technology, Mohali, Punjab 140306, India.
Biochem J. 2019 Aug 30;476(16):2411-2425. doi: 10.1042/BCJ20190453.
Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca-ions present in endolymph. Mutations at the Ca-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (10-10 pN s) as experienced in the inner ear. We performed network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.
纤毛顶端连接蛋白作为力感受器，在将声音产生的机械力传递至离子通道的同时，维持内耳中整个感觉装置的完整性。纤毛顶端连接蛋白的结构和功能之间的这种微妙平衡，是由内淋巴中的钙离子来调节的。顶端连接蛋白的钙离子结合位点发生突变，通常会导致先天性耳聋，有时还会导致伴有听力障碍的综合征缺陷。尽管已经发现了此类突变，但仍然不清楚突变如何改变与疾病相关的力感受器的结构-功能特性。为了从分子细节上阐明力感受器在传递力方面的差异，我们使用 FRET 技术，在接近内淋巴钙离子浓度的条件下，在单分子水平上鉴定了突变型和野生型顶端连接蛋白的构象可变性，随后使用原子力显微镜（AFM）的单分子力谱技术测量了力响应行为。AFM 使我们能够模拟内耳中经历的高幅度力斜坡（10-10 pN s）。我们进行了网络分析，发现突变体构象的改变会中断传感器的自然力传递路径，使突变型顶端连接蛋白容易受到声音刺激产生的输入力的影响。我们还证明，富含钙离子的环境可以恢复突变型顶端连接蛋白的力响应，这可能最终有助于设计出更好的治疗听力损失的策略。
