A Hydrophobic Bundle Crossing Found in Glutamate Receptor and K+ channel Pore Helices Dominates the Conformational Free Energy Landscape

Abstract

A conserved sequence of hydrophobic residues in ionotropic glutamate receptors is believed to be important for determining the channel's resting state. This sequence is homologous in many K+ channel helices, specifically the KcsA pore lining M2, for which a pH sensor controls channel gating. The mechanism of channel gating in KcsA remains poorly characterized. Umbrella sampling, a method to compute free energy differences based on MD simulations, is used to investigate the change in free energy required to transition from a closed to an open KcsA channel conformation. Physical mechanisms that control this transition are analyzed in detail using computational mutagenesis. A potential of mean force is constructed within a two-dimensional reaction coordinate between opposing helices in the M2 transmembrane region of KcsA. The M2 bundle crossing acts as a hydrophobic gate, providing 4 kcal/mol to the total 5.3 kcal/mol free energy barrier between a closed and open conformation. Two of the three known pH sensor residues E118 and E120 are important for stabilizing the intracellular region of the M2 bundle while the third, H25 on M1, initiates disruption of the hydrophobic gate initiating channel opening. Arginine residues within the charged intracellular region of M2 are also shown to stabilize the M2 bundle although do not drive M2 into an open channel conformation. Water density fluctuations at the hydrophobic gate boundary show vapor-liquid phase transitions as the channel pore opens. It is hypothesized that the M2 hydrophobic bundle crossing within KcsA provides a barrier to ion conduction through hydrophobic forces excluding water. H25 protonation provides the energy to disrupt the M2 hydrophobic gate inducing a local water phase transition. Hydration or wetting of the pore creates an open conducting ion channel.