Phosphorylation of Piezo1 at a Single Residue, Serine-1612, Regulates Its Mechanosensitivity and In Vivo Mechanotransduction Function

Neurology Basic Medical Sciences Biophysics Life Sciences Medicine Cell Biology Biochemistry Genetics
Piezo1 Serine-1612 Phosphorylation Mechanotransduction Endothelial Cells Blood Pressure Regulation

This article is a biomedical research paper authored by scholars such as Zhang Tingxin, Bi Cheng, and Li Yiran, published on November 6, 2024, in the journal “Neuron.” The research was led by a team from the Tsinghua University-Peking University Center for Life Sciences, exploring the regulatory mechanism of phosphorylation modification of the mechanosensitive calcium ion channel Piezo1 in physiological functions. The paper reveals how Piezo1 regulates its function through specific residue phosphorylation during the transduction of mechanosensitivity to achieve physiological effects on blood pressure homeostasis and exercise performance. This study fills the gap in the post-translational modification regulatory mechanism of Piezo1 channels and has potential clinical significance.

Background of the Study

Piezo1 and Piezo2 are known mechanosensitive cation channels that mediate the transduction process of mechanical forces in various cell types such as endothelial cells, red blood cells, osteoblasts, and cardiomyocytes. Piezo1, in particular, plays a key role in endothelial cells by sensing shear forces induced by blood flow to regulate vascular development, vascular tension, and blood pressure regulation. However, despite the widespread recognition of the physiological importance of Piezo channels in vivo, it remains unclear how Piezo1 channels achieve their mechanosensitivity regulation through post-translational modifications. This study focuses on how phosphorylation regulation of Piezo1 achieves precise control of mechanosensitivity and its transduction function.

Purpose of the Study

The primary goal of this study is to elucidate the phosphorylation sites of the Piezo1 channel and how phosphorylation affects Piezo1’s mechanosensitivity, particularly investigating the regulatory mechanism of PKA and PKC-mediated phosphorylation on Piezo1 function. The research team conducted a series of biochemical, molecular biology, and electrophysiology experiments in mice and in vitro cells to identify the key phosphorylation sites of Piezo1 and validate their physiological role in blood pressure and exercise endurance.

Research Methods

Experimental Procedures and Key Techniques

  1. Identification of Piezo1 Channel Phosphorylation Sites: The research team initially employed biochemical methods to determine a key site on Piezo1, specifically serine 1612 (S1612), as the main phosphorylation target of PKA and PKC. They used pull-down experiments and in vitro kinase assays with [γ-32P]-ATP to confirm that the Piezo1 protein is specifically phosphorylated in the presence of PKA or PKC.

  2. Electrophysiological Effects of Piezo1 Phosphorylation: Using in vitro electrophysiological testing of membrane currents in murine Piezo1, the research team found that activation of PKA with 8-Br-cAMP significantly increased the mechanosensitivity of the Piezo1 channel and significantly slowed the inactivation kinetics of the channel. The study suggests that phosphorylated Piezo1 exhibits higher mechanical sensitivity and stronger tolerance.

  3. Functional Verification of Piezo1 Channels in Endothelial Cells: Through primary culture of human umbilical vein endothelial cells (HUVECs), using the PKA agonist 8-Br-cAMP and adenylate cyclase activator forskolin, the research found that these compounds significantly enhanced the electrophysiological activity of the Piezo1 channel. Simultaneously, biotinylation and protein blotting experiments showed that PKA activation did not increase the expression of Piezo1 on the cell membrane.

  4. Construction and Functional Verification of Gene-Edited Mouse Models: Using CRISPR-Cas9 gene editing technology, a Piezo1-S1612A knock-in mouse model was constructed, mutating the site to a non-phosphorylatable form. In these knock-in mice, the study detected the functional loss caused by the mutation leading to Piezo1 phosphorylation deficiency, indicating that phosphorylation at the S1612 site is crucial for regulating the physiological function of the Piezo1 channel in vivo.

Research Data and Supporting Results

  1. Electrophysiological Results: After treatment with 8-Br-cAMP, the maximum current of the Piezo1 channel increased markedly from 1534 pA to 4090 pA, and the inactivation time increased from 23 ms to 69 ms, indicating that PKA activation increases Piezo1’s mechanosensitivity through S1612 phosphorylation.

  2. Physiological Experiments in Mice: Compared to normal mice, Piezo1-S1612A knock-in mice showed significantly elevated blood pressure at night and poor performance in exercise endurance tests, indicating that the loss of S1612 phosphorylation affects the mechanosensitivity regulation of the Piezo1 channel, which in turn affects blood pressure and exercise performance.

Conclusion

This study reveals a key phosphorylation site, S1612, on the Piezo1 channel, where phosphorylation by PKA and PKC effectively regulates the mechanosensitivity and inactivation properties of Piezo1, thus having significant physiological impacts on blood pressure homeostasis and exercise performance. This discovery not only aids in understanding the complex regulatory mechanisms of Piezo1 channels in vivo but also provides potential targets for future therapeutic strategies for related vascular diseases.

Significance of the Study

  1. Scientific Value: For the first time, the phosphorylation site of Piezo1 is identified, supplementing the structural-functional regulatory network of Piezo1 and providing new perspectives for exploring the physiological functions of Piezo1 in various cell types.

  2. Clinical Application Value: Since Piezo1 plays a role in various tissues such as vascular, nervous, and skeletal systems, this phosphorylation mechanism could become a novel molecular target for treating related diseases such as hypertension, cardiovascular diseases, and osteoporosis.

Highlights of the Study

  1. Discovery of the Regulatory Mechanism of the Key Phosphorylation Site S1612 on Piezo1: Phosphorylation changes the mechanical sensitivity of the channel, offering a new perspective for functional regulation of Piezo1.

  2. Construction of Piezo1-S1612A Knock-In Mice Using Gene Editing Technology: Validated the critical role of phosphorylation in the function of the Piezo1 channel, providing a unique model for exploring Piezo1’s physiological function in vivo.

  3. Elucidation of the Roles of PKA and PKC in Piezo1 Phosphorylation: Provides a scientific basis for future development of targeted drugs.

Additional Information

The study used a multi-level, multi-method approach to validate the importance of the phosphorylation site S1612, employing electrophysiology, proteomics, and gene editing technologies, ensuring high research quality. Additionally, experimental data and code can be provided upon request, with detailed experimental methods included, enhancing the transparency and reproducibility of the study.

This study demonstrates that the phosphorylation site S1612 on the Piezo1 channel plays a critical role in regulating blood pressure and exercise performance. The finding not only has significant value in understanding the physiological regulatory mechanisms of mechanosensitive channels but also may provide new targets for the treatment of related diseases, advancing research in the Piezo1-related field.