Abstract
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.
Highlights
The Piezo proteins are eukaryotic mechanosensitive cation channels that rapidly inactivate in a voltage dependent manner [1, 2] and are gated by membrane tension [3, 4]
We have previously shown that a fluorescent protein could be encoded in the hPIEZO1 protein without disrupting the properties of the channel [3]
The mean peak current is plotted as a function of the pressure (Fig 4B), and we determined the half-maximal pressure for activation (P1/2Boltzmann) to be 34 ± 0.9 mmHg (R2 = 0.99), slightly leftward shifted compared to wt at 40 ± 2.1 mmHg (R2 = 0.99) [12]
Summary
The Piezo proteins are eukaryotic mechanosensitive cation channels that rapidly inactivate in a voltage dependent manner [1, 2] and are gated by membrane tension [3, 4]. The C-terminal of the channel is necessary for the formation of the pore [7]. The physiological function of Piezo channels is currently under study. This protein was originally designated Fam38a and implicated in integrin activation and cell adhesion [8]. In Drosophila, Piezo was necessary for a rollover response of the larvae, and ablation of the gene reduced nociception [9]. Like Xerocytosis [10, 11] involve mutant Piezo
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