The activation thresholds and inactivation kinetics of poking-evoked PIEZO1 and PIEZO2 currents are sensitive to subtle variations in mechanical stimulation parameters

ABSTRACT

PIEZO1 and PIEZO2 are mechanically activated ion channels that confer mechanosensitivity to various cell types. PIEZO channels are commonly examined using the so-called poking technique, where currents are recorded in the whole-cell configuration of the patch-clamp technique, while the cell surface is mechanically stimulated with a small fire-polished patch pipette. Currently, there is no gold standard for mechanical stimulation, and therefore, stimulation protocols differ significantly between laboratories with regard to stimulation velocity, angle, and size of the stimulation probe. Here, we systematically examined the impact of variations in these three stimulation parameters on the outcomes of patch-clamp recordings of PIEZO1 and PIEZO2. We show that the inactivation kinetics of PIEZO1 and, to a lesser extent, of PIEZO2 change with the angle at which the probe that is used for mechanical stimulation is positioned and, even more prominently, with the size of its tip. Moreover, we found that the mechanical activation threshold of PIEZO2, but not PIEZO1, decreased with increasing stimulation speeds. Thus, our data show that two key outcome parameters of PIEZO-related patch-clamp studies are significantly affected by common variations in the mechanical stimulation protocols, which calls for caution when comparing data from different laboratories and highlights the need to establish a gold standard for mechanical stimulation to improve comparability and reproducibility of data obtained with the poking technique.

KEYWORDS:

• PIEZO1
• PIEZO2
• mechanotransduction
• poking technique
• patch-clamp
• mechanobiology

Acknowledgments

We would like to thank Mr Haider Al-Marsoomi for technical support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions statement

N.Z. – data acquisition, analysis, and interpretation of the data. S.G.L. – conception and design, analysis and interpretation of the data, and drafting of the paper. All authors approved the final version of the paper and agreed to be accountable for all aspects of the work.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by Deutsche Forschungsgemeinschaft under Grant LE3210/3-1.