Dissecting the Molecular Mechanism of How Force Activates Yeast TRP Channel TRPY1

Abstract

Though clear for the prokaryotic mechanosensitive (MS) MscL and MscS, how mechanical force activates eukaryotic MS channels remains poorly understood. Several members of transient receptor potential (TRP) channel family are apparently involved in animal mechanosensations and are therefore promising MS-channel candidates. The yeast homolog, TRPY1, expressed in the yeast vacuolar membrane, can clearly be gated by hyper-osmotic shock in vivo and by directly stretching excised vacuolar membrane patches under patch clamp. Here, we investigated the structure-function relationship of TRPY1 by mutagenesis, aiming at dissecting how force is sensed by TRPY1 and how force opens the channel gate. TRP channels share general organization with well-studied voltage-gated potassium channels, being tetramers with each subunit consisting of six transmembrane helices (S1-S6) and N- and C-terminal cytoplamic domains. We found that the C-terminal cytoplasmic domains of TRPY1 harbor Ca2+ binding motifs, which confer TRPY1 Ca2+ activation. We demonstrated that the Ca2+ activation and the force activation are synergistic and the two gating mechanisms act in parallel. We proposed that force is perceived by the transmembrane domains. Our further in-depth analyses showed that strategic insertions of long peptide linkers before S4-S5 linker and after S6 can surprisingly yield functional channels with largely intact mechanosensitivity, highlighting the crucial roles of the pore module in TRPY1 mechanosensitivity. Together with our detailed scanning mutagenesis, we will discuss possible molecular mechanisms on how force activates TRPY1.