Human PIEZO1: removing inactivation.

Area of Science:

• Biophysics
• Molecular Biology
• Cell Physiology

Background:

• PIEZO1 is a crucial mechanosensitive ion channel involved in cellular responses to mechanical stimuli.
• Inactivation of PIEZO1 is critical for its proper function, and its dysregulation is linked to xerocytotic anemia.
• Previous studies identified two sites influencing PIEZO1 inactivation, but complete removal of inactivation was not achieved.

Purpose of the Study:

• To engineer a PIEZO1 channel variant lacking inactivation by mutating specific sites.
• To investigate the functional consequences of eliminating PIEZO1 inactivation on channel gating and kinetics.
• To explore the interaction between mutation sites and their role in channel inactivation.

Main Methods:

• Site-directed mutagenesis was used to create a double mutant PIEZO1 (DhPIEZO1) with substitutions M2225R and R2456K.
• Electrophysiological recordings using complex stimulus waveforms were performed to analyze channel kinetics.
• Gating properties, including activation curves, deactivation rates, slope sensitivity, and unitary conductance, were measured.
• The effect of the gating modifier peptide GsMTx4 on DhPIEZO1 was assessed.

Main Results:

• The DhPIEZO1 double mutant exhibited a complete loss of inactivation.
• Channel activation shifted to lower pressures (∼30 mmHg), and deactivation rates slowed.
• Slope sensitivity and unitary conductance remained unchanged, indicating mutations do not affect pore properties or dimensional changes between states.
• GsMTx4 reversibly inhibited DhPIEZO1, acting as a gating modifier.
• Kinetic analysis revealed pressure-dependent rates for closing from the inactivated state and opening, with the energy barrier near the open state.
• Mutant cycle analysis demonstrated strong interaction between the two mutation sites.

Conclusions:

• Eliminating PIEZO1 inactivation via specific double mutations alters channel gating kinetics and pressure sensitivity.
• The identified mutation sites are critical for PIEZO1 inactivation and interact significantly, despite their proposed locations.
• Understanding PIEZO1 inactivation mechanisms provides insights into mechanotransduction and potential therapeutic targets for related disorders like xerocytotic anemia.