Protons in Gating the Kv1.2 Channel: A Calculated Set of Protonation States in Response to Polarization/Depolarization of the Channel, with the Complete Proposed Proton Path from Voltage Sensing Domain to Gate

Abstract

We have in the past proposed that proton motion constitutes the gating current in the potassium channel K_v1.2 and is responsible for the gating mechanism. For this to happen, there must be a proton path between the voltage-sensing domain (VSD) and the channel gate, and here we present quantum calculations that lead to a specific pair of proton paths, defined at the molecular level, with well-defined water molecule linkages, and with hydrogen bonding between residues; there is also at least one interpath crossover, where protons can switch paths. Quantum calculations on the entire 563-atom system give the complete geometry, the energy, and atomic charges. Calculations show that three specific residues (in the pdb 3Lut numbering, H418, E327, R326), and the T1 intracellular moiety, all of which have been shown experimentally to be involved in gating, would necessarily be protonated or deprotonated in the path between the VSD and the gate. Hydroxyl reorientation of serine and threonine residues are shown to provide a means of adjusting proton directions of motion. In the deprotonated state for K312, a low energy state, our calculations come close to reproducing the X-ray structure. The demonstration of the existence of a double proton path between VSD and gate supports the proposed proton gating mechanism; when combined with our earlier demonstration of proton generation in the VSD, and comparison with other systems that are known to move protons, we are close to achieving the definition of a complete gating mechanism in molecular detail. The coupling of the paths to the VSD, and to the PVPV section that essentially forms the gate, can be easily seen from the results of the calculation. The gate itself remains for further computations.

Keywords: amino acid strings; ion channel gating; proton transport paths.

Figure 1

General view of a K_v1.2 channel from the 3Lut coordinates. (A): View from the membrane (side view). Two of four domains are shown, with front and back domains deleted to allow the structure to be seen. (B): A 90° rotation of the channel, showing the pore. In neither (A) nor (B) are individual amino acids shown. The T1 section is somewhat visible in (A). Neither (A) nor (B) make it clear that the linker is not entirely continuous unless the water is included, although water is not shown in these figures. (C): A single domain is shown, making the domain crossing clear. Although some individual amino acids are shown with specific colors, it is too difficult in this view to see the relation of the actual amino acids, and again the water is not shown. The region that was calculated is labeled here as linker and T1, although it is hard to see the exact boundary in this type of figure. Figure 2, Figure 3 and Figure 4 show the specific calculations, with individual amino acids and water explicit and clear.

Figure 2

This figure shows two views (system rotated to make it possible to see all the residues clearly) of the results of the optimization with protons on the almost adjacent residues K312 and R326. Key residues are labeled in (B), which is the same as (A) but rotated 90°. The energy for this result turns out to be more than 30 k_bT above the other two cases. The two charged residues separate by more than 2 Å beyond the X-ray value. Both proton paths are easily visible, by following the colored residues, and the connecting (meaning hydrogen bonded) water molecules. The upper path includes K312 and R326, and, in this optimization, ends at N412, from which it has access to the conserved PVPV sequence. The lower path, which includes Y415, continues via H418 to N414, which likewise has access to the PVPV sequence. Other amino acids included in the optimization, but not the proton path, are shown in gray. In addition to the upper and lower paths, there is a crossover that apparently allows protons to switch paths; this is the vertical path in the left-center. The gate is on the left, the VSD on the right and above. Color code: Carbon, green; nitrogen, large blue sphere; hydrogen, small pale blue spheres; oxygen, magenta. The added protons are not visible, but their positions are indicated by the arrows.

Figure 3

As in Figure 2, the key residues are labeled in (B), and the two paths, plus the crossover, can be seen by following the colored residues. The color code is the same, and the arrows again indicate the position of the two mobile protons (in (A) R326 is indicated; that residue is always protonated, but is indicated here as well). Rotation from (A) to (B) is as in Figure 2. This is one of the two low energy configurations, so this arrangement is considered to be part of the actual pathway.

Figure 4

This is the other low energy configuration. All conventions (color code, rotation, arrows for protons) are as in Figure 2 and Figure 3. This case is similar to the X-ray structure. (A,B) figures correspond to the (A,B) of Figure 2 and Figure 3; (B) contains the labels on specific amino acids.

Figure 5

This figure is taken directly from the 3Lut coordinates, and shows the relation of the calculated section, principally the upper path, to the gate and the VSD. Upper path residues are labeled; all else that is included is in gray. R309 and H310 are not part of the calculation, but form the link to the VSD. All four domains at the gate are shown in part, so that the orientation of the linker can be understood.