Mechanotransduction is the process by which mechanical stimuli are converted into biological activity. Piezos are mechanically activated (MA) cation channels conserved through evolution and act as mechanotransducers in various biological processes. The single Piezo gene in flies is involved in nociception1; zebrafish and mouse Piezo2, in touch sensation2-6; zebrafish Piezo1, in red blood cell volume regulation7; and mouse Piezo1, in vascular development8, 9. In humans, mutations that alter channel gating of Piezo1 and 2 are linked to various disorders with dominant inheritance10-12. Piezo proteins contain over 2000 amino acid residues with an estimated 30 to 40 transmembrane (TM) segments and are likely to form homo-tetramers in a complex weighing over 1.2 million daltons13, 14. Piezos lack homology with other proteins, and their structural features remain unknown. The large size and numerous hydrophobic domains of Piezos constitute technical challenges for structural analysis of the intact channel15. Basic questions regarding Piezo topology and the location of the ion permeation pathway remain unanswered, and yet these questions are crucial for a mechanistic understanding of how the channel is gated by mechanical forces, and how human disease-related point mutations affect channel function16. We used a combination of bioinformatics and detection of inserted tags and phosphorylated sites to study mouse Piezo1 transmembrane topology. Using a chimeric approach followed by site directed mutagenesis, we identified a residue that when mutated impacts ion permeation properties. We propose that this amino acid is either in the pore or closely associates with the pore.