Functional characterization of native Piezo1 as calcium and magnesium influx pathway in human myeloid leukemia cells

Piezo1 is a Ca²⁺-permeable mechanically activated ion channel that is involved in various physiological processes and cellular responses to mechanical stimuli. The study of biophysical characteristics of Piezo1 is important for understanding the mechanisms of its function and regulation. Stretch activation, a routine approach that is applied to stimulate Piezo1 activity in the plasma membrane, has a number of significant limitations that complicate precise single-channel analysis. Here, we aimed to determine pore properties of native Piezo1, specifically to examine permeation for physiologically relevant signaling divalent ions (calcium and magnesium) in human myeloid leukemia K562 cells using Piezo1-specific chemical agonist, Yoda1. Using a combination of low-noise single-current patch-clamp recordings of Piezo1 activity in response to Yoda1, we have determined single-channel characteristics of native Piezo1 under various ionic conditions. Whole-cell assay allowed us to directly measure Piezo1 single currents carried by Ca²⁺ or Mg²⁺ ions in the absence of other permeable cations in the extracellular solutions; unitary conductance values estimated at various concentrations of Mg²⁺ revealed strong saturation effect. Patch clamp data complemented with fluorescent imaging clearly evidenced Ca²⁺ and Mg²⁺ entry via native Piezo1 channel in human leukemia K562 cells. Mg²⁺ influx via Piezo1 was detected under quasi-physiological conditions, thus showing that Piezo1 channels could potentially provide the physiological relevant pathway for Mg²⁺ ion transport and contribute to the regulation of Mg²⁺-dependent intracellular signaling.