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PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. [ABSTRACT FROM AUTHOR]

Abstract: Sensing the energy from mechanical inputs is ubiquitous––and perhaps the oldest form of biological energy transduction. However, the tools available to probe the mechanisms of transduction are far fewer than for the chemical and electric field sensitive transducers. The one pharmacological tool available for mechansensitive ion channels (MSCs) is a peptide (GsMTx-4) isolated from venom of the tarantula, Grammostola spatulata, that blocks cationic MSCs found in non-specialized eukaryotic tissues. In this review, we summarize the current knowledge of GsMTx-4, and discuss the inevitable crosstalk between the MSC behavior and the mechanical properties of the cell cortex. [Copyright &y& Elsevier]