Mechanism of activation at the selectivity filter of the KcsA K+ channel
- University of Basel, Switzerland
- Weill Cornell Medical College, United States
Figures
Figure 1 with 4 supplements
Potential of mean force calculations describing ion permeation through the selectivity filter in the closed and open intracellular gate conformations of the channel.
(A) In the closed conformation (based on structure 1K4C), ions are tightly bound to the selectivity filter in states S1-S3-S5 or S0-S2-S4 with little ion movement due to high free energy barriers > 6…
Figure 1—figure supplement 1
Potential of mean force describing ion permeation in the selectivity filter of the closed KcsA channel starting from the S1-S3-Cav occupancy state.
When an ion moves from the cavity to the S4 binding site, no water molecule is seen between this ion and the one in S3. The S4 binding site seems to be too small in volume and not flexible enough to …
Figure 1—figure supplement 2
Modeling of the open-activated selectivity filter based on open-inactivated structures.
(A) A network of water molecules found around the permeation pore stabilizes the collapsed, inactivated state of the selectivity filter. Water molecules bridge the subunits at the level of residues …
Figure 1—figure supplement 3
Potential of mean force describing ion permeation in the open channel.
The free energy map on the left is identical to the one presented in Figure 1B and describes the recruitment by the selectivity filter of an ion coming from the cavity. The map on the right …
Figure 1—figure supplement 4
Standard deviation for the PMFs presented in Figure 1.
Each standard deviation map is calculated from an ensemble of five PMFs calculated on intervals of 100 ps.
Figure 2
Fluctuations around the selectivity filter.
(A) The histogram shows the inter-subunit, adjacent (1) and opposite (2), distances between the Cα atoms of residues 75 to 78. In the open state of the channel (two independent simulations shown in …
Figure 3
Contacts between the TM helices and the pore helix that can potentially transmit the activation signal.
(A) vdW interactions of the individual residues of the TM1 and TM2 helices with the pore helix. Residue Leu40 from TM1 forms the strongest interactions. Interactions involving Ser44 are also …
Figure 4
Coupling of the selectivity filter to the intracellular gate via residue L40.
(A) The pH sensor residues, H25 and E118, are found on the intracellular side of the channel. Residues H25 and L40 both lie on the transmembrane helix TM1. (B) KcsA L40A open probability (Po) vs. pH …
Figure 5 with 5 supplements
Impact of the L40A mutation on ion permeation in the closed and partially activated channel.
(A) The PMF calculations show that when the intracellular gate is closed (pdb entry 1K4C), ion diffusion is impeded in the control channel (E71A) with a free energy difference between the 3-ion …
Figure 5—figure supplement 1
Forward and backward PMFs of the transition between the S1-S3-Cav and S0-S2-S4 ion occupancy states in the control and L40A mutant channels for (A) the closed (1K4C) and (B) the partially activated (3F7V) conformations.
The forward transition, from S1-S3-Cav to S0-S2-S4, is plotted on the left. The starting occupancy state is stated in parentheses (S1-S3-Cav) and identified by a star on the plots. On the right is …
Figure 5—figure supplement 2
Potential of mean force calculation describing ion permeation in the E71A mutant in the fully open conformation (pdb entry 3F5W).
The PMF is presented as in Figure 1.
Figure 5—figure supplement 3
Ion permeation in the partially activated channel with the L40A mutation.
Ion trajectories are shown for a simulation of the E71A/L40A double mutant in the partially activated conformation (pdb entry 3F7V) using a transmembrane voltage of 400 mV and an ion concentration …
Figure 5—figure supplement 4
Convergence of the potential of mean force calculations.
(A, B) The sampling for the forward (initial state: S1-S3-cav) PMFs based on the partially activated structure (pdb entry 3F7V) presented in Figure 5—figure supplement 1 has been split in 100 ps …
Figure 5—figure supplement 5
Standard deviation for the PMFs presented in Figure 5—figure supplement 1.
Each standard deviation map is calculated from an ensemble of five PMFs calculated on intervals of 100 ps.
Figure 6
The selectivity filter as the center of the activation mechanism.
(A) The superposition of the open and closed channel illustrates how the movement of the TM1 helices (arrow 1) allows for the displacement of the pore helix and slight expansion of the selectivity …
Tables
Table 1
Summary of Hill and pH-gating model fits for the KcsA mutants indicated.
https://doi.org/10.7554/eLife.25844.011| Mutant* | Hill fit | pH sensor 1 (H25) | pH sensor 2 (E118) | Intrinsic gating | |||
|---|---|---|---|---|---|---|---|
| pH1/2 (±) | nH (±) | pKa1closed (±) | pKa1open (±) | pKa2closed (±) | pKa2open (±) | Lo (±) | |
| E71A† control channel | 5.3 (0.01) | 4.4 (0.1) | 4.8 (N/A)‡ | 7.6 | 5.0 | 6.2 | 2.5E-12 (0.3E-12) |
| L40A | 5.2 (0.02) | 1.9 (0.2) | 7.6§ | 7.6 | 5.0 | 6.2 | 6.2E-4 (0.7E-4) |
| H25R† | 5.3 (0.02) | 1.9 (0.2) | - | - | 5.0 (0.1) | 6.2 (0.1) | 7E-4 (4E-4) |
| E118A† | 5.5 (0.03) | 4.5 (2.8) | 4.8 | 7.6 | - | - | 1.1E-8 (0.1E-8) |
-
*All mutants on the background of E71A.
†Data and fits from Refs. (Thompson et al., 2008) and (Posson et al., 2013a).
-
‡Not applicable; no error given when the parameters were constrained (see Materials and methods).
§The data were fit by abolishing the H25 pKa state-dependence, similar to the H25R mutant.
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.25844.019
