Abstract
Piezo1 and Piezo2 encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. Structural features of Piezos remain unknown. Mouse Piezo1 is bioinformatically predicted to have 30–40 transmembrane (TM) domains. Here, we find that nine of the putative inter-transmembrane regions are accessible from the extracellular side. We use chimeras between mPiezo1 and dPiezo to show that ion-permeation properties are conferred by C-terminal region. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Our results describe important structural motifs of this channel family and lay the groundwork for a mechanistic understanding of how Piezos are mechanically gated and conduct ions.
Highlights
Piezo[1] and Piezo[2] encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively
To characterize the transmembrane topology of mPiezo[1] we combined bioinformatics analysis, immunostaining to detect extracellular tags inserted in predicted loop regions, and detection of intracellular phosphorylation sites by mass spectrometry (Fig. 1)
Extracellular Myc tag Phosphorylated site Myc tag detected only after permeabilization comparable to dPiezo (Fig. 2 and Supplementary Table 3). These results suggest that domain(s) required for determining conductance and pore block are encoded by the region of the protein sequence starting from mPiezo[1] amino acids 1974
Summary
Piezo[1] and Piezo[2] encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Combining Myc-immunostaining and phosphorylation sites together with the bioinformatics analysis allowed us to construct a model topology, which predicts 38 transmembrane domains with intracellular N- and C-termini (Fig. 1b). This extreme scenario would predict a topology with a minimum of 10 TM domains
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