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PIEZO1 and PIEZO2 are mechanosensitive channels (MSCs) important for cellular function and mutations in them lead to human disorders. We examined how functional heteromers form between subunits of PIEZO1 using the mutants E2117K, E2117D, and E2117A. Homomers of E2117K do not conduct. E2117A homomers have low conductance with rapid inactivation, and those of E2117D have high conductance with slow inactivation. Pairing E2117K with E2117D or E2117A with E2117D gave rise to new channel species representing heteromers with distinct conductances. Whole-cell currents from co-expression of E2117A and E2117D fit well with a linear-combination model of homomeric channel currents suggesting that functional channels do not form from freely-diffusing, randomly-mixed monomers in-vitro. Whole-cell current from coexpressed PIEZO1/PIEZO2 also fit as a linear combination of homomer currents. High-resolution optical images of fluorescently-tagged channels support this interpretation because coexpressed subunits segregate into discrete domains. [ABSTRACT FROM AUTHOR]

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]