Probing the Energy Landscape of Activation Gating of the Bacterial Potassium Channel KcsA

Abstract

The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating.

Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs

“…Initial tests using the adenosine A 2A receptor and β 2 adrenergic receptor suggested that this method could reliably predict the orientation of the active TM6 position based on an inactive starting structure (data not shown). The best scoring active and inactive models were used as starting conformations for essential dynamics simulation [33] . Each protein was embedded in an equilibrated solvated membrane consisting of 280 POPC lipids.…”

Molecular Dynamics Simulations of KirBac1.1 Mutants Reveal Global Gating Changes of Kir Channels

“…It is conceivable that an energetic barrier needs to be overcome in WT for channel opening as shown in a previous simulation study on KcsA. 41 …”

“…Potential of mean force (PMF) calculations were performed as described previously. 41 Briefly, the main conformational changes in the most prominent G143E d opening simulation were obtained by principal component analysis. The first eigenvector obtained by this PCA was used as a reaction coordinate.…”

Probing Allosteric Coupling of a Constitutively Open Mutant of the Ion Channel KcsA using Solid State NMR

“…Potassium channels typically contain a selectivity filter, which selects for 4 potassium over other ambient ions with high affinity, as well as an activation 'gate' that 5 opens the channel and initiates the flow of potassium. In many potassium channels, 6 however, the channel spontaneously stops conduction even when potassium ions and hypothesized that the site of inactivation is in the selectivity filter, where potassium 18 ions are bound and conducted through the membrane [2,9,23,40]. This mechanism is 19 termed activation-coupled inactivation, i.e., and proposes that an allosteric coupling 20 between the selectivity filter and activation gate (like the pH gate in KcsA) lead to a 21 decrease in potassium affinity at the selectivity filter after activation.…”

“…This mechanism is 19 termed activation-coupled inactivation, i.e., and proposes that an allosteric coupling 20 between the selectivity filter and activation gate (like the pH gate in KcsA) lead to a 21 decrease in potassium affinity at the selectivity filter after activation. A direct 22 measurement of the potassium ion affinity at the selectivity filter by solid state nuclear 23 magnetic resonance (SSNMR) was carried out on KcsA embedded in an authentic lipid 24 environment [42]. The potassium ion affinity changed from K apparent = 14 ± 1 mM at 25 pH 3.5 to 4 ± 1 µM at pH 7.5.…”