Molecular Dynamics Studies of Permeation and Gating Mechanisms in Potassium Channels

Abstract

Potassium (K+) channels contribute to diverse physiological activities, representing an indispensable part of living organisms. A thorough understanding of different aspects of permeation mechanisms and gating in K+ channels is of crucial importance. Since only static information can be inferred from the available high-resolution structures of K+ channels, the dynamical nature of ion permeation calls for a systematic investigation into the collective behavior of ions during permeation using computer simulations. In the first part of the thesis, we use molecular dynamics simulations and Markov state models to obtain dynamical details of multi-ion permeation processes in the selectivity filter. The permeation cycles representing ion permeation events reveal the dominance of the water-free direct knock-on permeation mechanism over a wide range of salt concentration, temperature, and transmembrane voltage for MthK. Other potassium channels with a highly conserved selectivity filter also demonstrate direct knock-on. Finally, charge strength dependence of permeation cycles was explored. The results illustrate the robustness of direct knock-on and shed light on the dynamical aspect of details in ion conduction, providing valuable insights into the conduction mechanisms in potassium channels. The second part of the thesis delves into the possibility of channel gating via switching between two conformations. TREK-2 responds to a wide range of stimuli, such as pH, membrane tension, and binding of small ligands. The crystallographic up and down conformations have been resolved, where their main difference lies in the position and orientation of the transmembrane helix M4. However, molecular explanations for gating mechanisms and whether transitions between these two conformations play an essential role in gating remain elusive. Here, we use molecular dynamics simulations and free energy calculations to determine how the equilibrium between the up and the down conformations of TREK-2 is shifted by mutations. Mutants that exhibit considerable shifts away from the down state are identified. Most conformational shifts due to mutations can be attributed to induced steric clashes or weakened favorable interactions in the cytoplasmic part of M2, M3, and M4. These findings are in excellent agreement with functional analysis and NFx inhibition measurement of TREK-1 WT and mutants, unraveling the molecular roles of the identified residues in governing transitions between the two conformations and suggesting that conformational switching between the up and down states is a viable gating mechanism for TREK-1 and TREK-2.