The OSCA family proteins are pivotal mechanosensitive ion channels in plants, playing a crucial role in sensing osmotic stress and initiating drought adaptation pathways. Despite significant progress in structural biology, the dynamic conformational pathway of gating, the cooperative mechanism of key residues, and the mechanism of selective permeation for different cations remain largely elusive. In this study, we employed molecular dynamics (MD) simulations to systematically investigate the Arabidopsis OSCA3.1 channel in its contracted, expanded, and mutant open states. Conventional MD simulations reveal that the transition from the contracted to the open state is accompanied by a significant conformational rearrangement involving the tilting of the M6 helix. This movement drives the radial displacement and reorientation of key hydrophobic residues (e.g., F504, F505), thereby dismantling the hydrophobic gate composed of continuous hydrophobic residues and creating physical space for ion permeation. Steered molecular dynamics (SMD) simulations show that both K+ and Ca2+ must overcome substantial resistance when traversing the hydrophobic constriction. However, due to its higher charge density and dehydration energy, Ca2+ requires a significantly larger driving force than K+. Umbrella sampling calculations yielded potential of mean force (PMF) profiles, quantitatively revealing the free energy barrier differences for the permeation of the two ions for the first time. The results indicate that although their permeation pathways are similar, Ca2+ faces a significantly higher free energy barrier than K+ (Ca2+: ∼53 kcal/mol vs K+: ∼30 kcal/mol) due to stronger hydration and the higher energy cost of dehydration associated with its higher charge density. Electrostatic potential analysis further shows that the hydrophobic gating region within the pore is electrically neutral, facilitating the passage of various cations, while the lower part is electronegative, a distribution that may favor initial ion entry and subsequent guidance. This study demonstrates that the opening of the OSCA3.1 channel is a highly cooperative conformational reorganization process dominated by helical motion. Although its internal environment allows for the passage of multiple cations, from an energetic perspective, the conduction of monovalent K+ is more favorable in this expanded state. These findings demonstrate that OSCA proteins exhibit a "kinetic selectivity" rather than being "completely nonselective," where mechanical force or osmotic pressure modulates the degree of channel opening, thereby dynamically affecting the selective transport efficiency of potassium and calcium ions. This provides a novel atomistic mechanism and energetic theoretical basis for understanding how plants differentially regulate the transmembrane flux of the osmotic balance ion (K+) and the signaling ion (Ca2+) through the same channel when sensing osmotic stress.
Xiong et al. (2026) studied this question.