Mechanism of Mechanosensitive Gating of the TREK-2 Potassium Channel

Abstract

The mechanism of mechanosensitive gating of ion channels underlies many physiological processes, including the sensations of touch, hearing, and pain perception. TREK-2 is the best-studied mechanosensitive member of the two-pore domain potassium channel family. Apart from pressure sensing, it responds to a diverse range of stimuli. Two states, termed "up" and "down," are known from x-ray structural crystallographic studies and have been suggested to differ in conductance. However, the structural details of the gating behavior are largely unknown. In this work, we used molecular dynamics simulations to study the conductance of the states as well as the effect of mechanical membrane stretch on the channel. We find that the down state is less conductive than the up state. The introduction of membrane stretch in the simulations shifts the state of the channel toward an up configuration, independent of the starting configuration, and also increases its conductance. The correlation of the selectivity filter state and the conductance supports a model in which the selectivity filter gates by a carbonyl flip. This gate is stabilized by the pore helices. We suggest a modulation of these helices by an interface to the transmembrane helices. Membrane pressure changes the conformation of the transmembrane helices directly and consequently also influences the channel conductance.

Figure 1

Crystallographic difference. Shown is the structural difference of the crystal up (PDB: 4BW5, x-ray up) and down (PDB: 4XDJ, x-ray down) structures of TREK-2 without C-terminus (19). The major difference between the two configurations can be found in the lower helix bundle, whereas the configuration of the selectivity filter is indistinguishable (inset). On the top of the channel, a cap region is located. The membrane in which the channel is embedded is sketched in the background. To see this figure in color, go online.

Figure 2

Permeations and carbonyl flips. (A) The conformation of the selectivity filter is shown on the left. S1–S4 correspond to the carbonyls separating the binding sites rather than to the binding sites themselves. The selectivity filter is filled with potassium ions (spheres). The carbonyl S3 shows a flipped configuration with a water molecule inside the selectivity filter. (B) Dots represent ion permeation events. In relation to this, the selectivity filter configuration is shown as lines (Flip S1–S4 represent the state of the corresponding carbonyl group). If the line is at the bottom, it represents a configuration in which all carbonyls of the corresponding site of the selectivity filter are in their canonical configuration (as seen in Fig. 2A for S2). The top represents a state in which at least one carbonyl of the respective site is steadily flipped (as seen in Fig. 2A for S3). If the line is in the intermediate area, it represents a temporarily flipped state (less than 1 ns), and the height shows its duration. If no flip is present, the channel shows permeation events. When S3 flips after around 150 ns, permeations stop. At around 400 ns, the S3 carbonyl flips back into the canonical configuration, and permeations resume. This behavior is representative for all simulations. To see this figure in color, go online.

Figure 3

Effects of membrane tension on the up-down difference vector and on the TM2 helix angle. (A) Shown is a projection of simulations at different membrane tensions projected onto the difference vector of the crystallographic states (excluding the cap). On the x axis, the membrane tension is shown. The figure at the bottom represents the simulation setup where the channel is located inside the membrane and is shown with some ions surrounding them. The arrows represent the stretch applied to the membrane. The lines in (A) represent the configuration of the crystallographic state (x-ray down, PDB: 4XDJ; x-ray up, PDB: 4BW5). The violins represent the probability of the simulations starting from the up and down structures to be at the position on the difference vector. The dot in the violin represents the average over all simulations, and the bars represent the variance of the data points summarized in the respective violins. MD simulations using the CHARMM force field are highlighted. Going from a negative (compression) to a positive (stretch) tension of the membrane, the configuration of TREK-2 moves along the difference vector from a more down-like configuration to a more up-like configuration. Only the CHARMM simulations are flexible enough to reach the up state. (B) Quantification of the helix angle between the transmembrane helix 2 (TM2) of the two subunits. The violins show the distribution of the helix angle for different membrane tensions. The helix flattens out in the membrane with stretch applied (represented by a larger TM2 angle between the helices), whereas a compression of the membrane results in a steeper position of the helix in the membrane, which is represented by a smaller angle between the TM2 helices of the subunits. Up and down simulations show a minor difference, with the up simulations having a tendency toward a larger helix angle. deg, degree. To see this figure in color, go online.

Figure 4

Relation of up- and down state to conductance. Here, we summarize our results into a gating model. The down state is more likely to be in a nonconductive conformation than the up state (lines). The down state is represented by a selectivity filter configuration with a flip and a water molecule in the selectivity filter. In contrast, the up state is more likely to be in a conductive configuration, as represented by a canonical selectivity filter with full ion occupancy. Applying membrane tension (arrows) changes the state of the protein toward a more up-like configuration that is more conductive. To see this figure in color, go online.