| dc.description.abstracteng | ABSTRACT
Mechanosensation is an integral property of sensory systems like audition and somatosensation. The molecular determinants of mechanotransduction in mammals are only beginning to be understood. In 2010, Piezo2, a member of the Piezo family, was identified as the bona fide mechanotransducer in the somatosensory system of mice. Since then Piezo2 has been implicated in light touch, proprioception, mechanical allodynia and hyperalgesia. In order to understand more about the function of Piezo channels and their role in mechanosensation, an understanding of its regulators is vital. Protein-protein interactions are the bedrock of ion channel regulation and in the context of Piezo2, few such interactions (Stoml3 and Runx1) have been described. Recently it has also been found that the activity of Piezo2 is linked strongly to the properties of the cellular membrane. Depletion of cholesterol, mediated by Piezo2 interactor Stoml3, was shown to potentiate Piezo2 mediated currents. Furthermore, TRPV1 mediated modulation of Piezo2 was shown to involve membrane phospholipid PI(4,5)P2.
In 2016, our lab reported the first systematic characterization of the Piezo2 interactors in somatosensory neurons of mice. Through a mass-spectrometry based interactomics approach, 36 proteins were identified as potential Piezo2 interactors. Within these proteins we found some known modulators of somatosensory mechanotransduction including Kv1.1 and CamKII. Besides these, there were some proteins which presented as highly unlikely interactors of Piezo2, such as Pericentrin (Pcnt). Other proteins identified in the screen were involved in cellular processes like protein localization, protein phosphorylation and vesicle mediated transport.
In the current study, I describe the characterization of two of the Piezo2 interactors identified in the mass-spectrometry screen. The first part of the results describes Pericentrin (Pcnt) as a low probability interactor of Piezo2. The results show that Pcnt is expressed in close proximity with Piezo2 in both peripheral endings and cell bodies of DRG neurons. Additionally, Pcnt is shown to functionally modulate Piezo2 in DRG neurons. Loss of Pcnt in these neurons was found to potentiate Piezo2 currents and increase Piezo2 membrane expression. Though the mechanism of Piezo2-Pcnt interaction remains to be explored, the results do provide validation of the interactomics screen. The fact that the screen can successfully identify bona fide Piezo2 interactors makes it a useful resource for understanding Piezo2 function.
The second part of the results aims to characterize the interaction between Piezo2 and Mtmr2. Mtmr2, is a member of the myotubularin phosphatase family which is involved in the phospholipid synthesis pathway. It was identified as a high probability interactor of Piezo2, and notably, two other members of the same family, Mtmr1 and Mtmr5 (Sbf2) were also identified in the screen. The results presented here suggest that Mtmr2 is involved in a bi-directional regulation of Piezo2 function in DRG neurons i.e. loss of Mtmr2 potentiates Piezo2 mediated currents and overexpression of Mtmr2 suppresses Piezo2 currents. Furthermore, the phosphatase activity of Mtmr2 was found to be indispensable for its regulation of Piezo2. Pharmacological modulations of the phospholipid pathway show that PI(3,5)P2 plays a crucial role in the regulation of Piezo2. Further evidence for phospholipid interaction with Piezo2 came with the identification of a peptide segment of Piezo2 that can bind to PI(3,5)P2. Additionally, modulation of Piezo2 by osmotic stress is shown to rely on PI(3,5)P2 levels and Mtmr2 activity. The results presented here provide additional evidence for regulation of Piezo2 by membrane lipids and how proteins regulating these lipids are relevant to Piezo2 physiology.
In summary, since the identification of Piezo2, a quest to learn about its function and regulation has been underway. The results of this study combined with the interactomics screen published in 2016 have attempted to answer some fundamental questions about Piezo2 physiology. A characterization of the other proteins identified in our screen will help reveal more facets of Piezo2 regulation. Future studies will also add to structural information on Piezo2 which can further help elucidate its role as the bona fide mechanotransducer of the mammalian somatosensory system. | de |