Gating crosstalk in potassium channels

Figures

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  Fig. 1.. Structure of the pore domain of a K+ channel. (A) Open (cyan) and closed (white) structures of MthK. Two subunits are shown for clarity. The four K+ binding sites at the selectivity filter are labeled as S1–S4. The Cα atoms of the conserved glycine (G83) in the inner transmembrane helices are shown as spheres. The pore-lining hydrophobic residue (I84), the threonine (T59), and the threonine Oγ–Oγ distance are also indicated. (B) Experimentally observed structural changes in the selectivity filter during C-type inactivation, including constriction, dilation, carbonyl flip, and subtle changes in the size of a K+ binding site. (C) Schematic representation of the key structural elements involved in the proposed gating crosstalk model. We highlight coupling between the movements of the hydrophobic residue from the inner helix and the threonine at the filter entrance.

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  Fig. 2.. Structural overview of the six examined K+ channels. Two subunits are shown for each channel for clarity. All experimentally determined domain structures are displayed to illustrate the structural diversity of the channels. The PDB entries shown are 6U6D (MthK) (32), 5U76 (cBK) (33), 4XDJ (TREK-2) (34), 3EFF (KcsA) (35), 7F0J (Kv4.2) (36), and 5TJI (aBK) (37). For illustration purposes, for KcsA and Kv4.2, structures different from those used in the simulations (see the Methods) are shown here to display their intracellular regions. For MthK, cBK, KcsA, Kv4.2, and aBK, only the pore domain (which is shown in gray) was included in the simulations, whereas the extracellular domain was also retained for the simulations of TREK-2. See SI Appendix, Fig. S1 for a detailed comparison of the pore domains used in simulations of these channels. The conserved glycine (or proline) is also indicated.

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  Fig. 3.. Cavity hydration level and filter entrance size are coupled to regulate ion permeation. (A) Strategy for controlling the conformation of the inner transmembrane helices in MthK and cBK. We apply restraints either to maintain the disruption of the backbone H-bonds at the kink or to promote their reformation. See SI Appendix section 2.1.3 and Fig. S11E for detailed restraint protocols. (B) The ion current, the width of selectivity filter entrance (characterized by the threonine Oγ–Oγ distance, particularly T59 in MthK, T293 in cBK, and T172 and T281 in TREK-2) and the hydration level of the central cavity (number of waters in the cavity) in the simulations of MthK, cBK, and TREK-2. For each channel, we compared the results of different structures (C: closed; O: open; 1-4Bent of MthK: promote reformation of backbone H-bonds in different numbers of subunits; O.Kinked or O.Bent of cBK: maintain the disruption of H-bonds or promote their reformation, respectively; O.Kinked.Res and O.Bent.Res: restraining the width of the filter entrance in addition to H-bonds; see panel A). Data are presented as boxplots. The center line, box limits, and whiskers represent the median, upper and lower…

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  Fig. 4.. A hydrophobic residue at the kink affects the width of the selectivity filter entrance. (A) The identified hydrophobic residues (Hyd) in each channel. The selectivity filter is shown as stick model. The hydrophobic residues are shown as space filling model and colored based on Hyd–Thr contact probability. The values of the contact probability in different states are noted below each channel (C: closed; O: open; Par.O: partially open; Bent: promote backbone H-bond reformation; Kinked: maintain H-bond disruption; C.Mut in aBK: F304A/I308A mutant in the closed state, 10 parallel simulations are classified into two groups based on their Oγ–Oγ distances, called C.Mut.g1 and C.Mut.g2). (B) The Thr–Thr and Hyd–Hyd distances. We show TREK-2 as an example. The Thr–Thr distance is defined as the distance between the center of mass of the threonine side chains from opposite subunits. The Hyd–Hyd distance is defined analogously. (C) Pearson correlation coefficient (PCC) between the Thr–Thr and the Hyd–Hyd distance. The error bars represent SEM. The symbol size is larger than the error bar in most cases. (D) Correlation between the PCC values and the Hyd–Thr contact probabilities. (E)…

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  Fig. 5.. Experimentally derived structures in the Protein Data Bank reveal distinct Hyd–Hyd and Thr–Thr distances between open and closed states. We compare the open and closed states of MthK (m = 13, n = 7), NaK2K (m = 56, n = 7), aBK (m = 6, n = 5), TREK-2 (m = 3, n = 5), and Kv4.2 (m = 10, n = 4). Numbers in parentheses (m, n) denote the numbers of available open and closed structures. Note that for Kv4.2, the hydrophobic residue is V395 in the open state and I398 in the closed state. We highlight this change by using solid dots (instead of open circles) to show the data calculated from its open structures (also see SI Appendix, Fig. S6D). The statistics of open and closed states are shown as boxplots. The center line, box limits, and whiskers represent the median, upper and lower quartiles, and 1.5× interquartile range. The averages are shown as solid dots or open circles in red and black.

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  Fig. 6.. The filter entrance size modulates ion occupancy at the S4 site. (A) Schematic illustration of the proposed mechanism by which the filter entrance size affects ion permeation rate. (B) Dependence of the Oγ–Oγ distance distribution on the S4 occupancy state. We show results of the open states of MthK and cBK. See SI Appendix, Fig. S7A for results of other channels.

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  Fig. 7.. Larger selectivity filter entrance width promotes carbonyl flips. (A) Carbonyl flip at the S3 site. Water molecules behind the selectivity filter may stabilize flips through hydrogen bonds. (B) Carbonyl flip probability presented as boxplots. Top panel: probabilities at individual ion binding sites (S1–S4), pooled across simulations of all channels. Bottom panel: probabilities for individual channels. The center line, box limits, and whiskers represent the median, upper and lower quartiles, and 1.5× interquartile range. The averages are shown as red dots. Each open gray circle represents the average of a group of parallel simulations. (C) Flip probability as a function of the Oγ–Oγ distance in cBK, TREK-2, and aBK. Each data point represents the average of a group of parallel simulations, with error bars indicating the SEM. (D) Long-lived carbonyl flip inhibits ion permeation. A representative simulation of cBK is shown. Purple indicates ion permeation events, blue indicates water permeation events and water occupancy at S2 and S3 sites, and orange indicates carbonyl flips at S3.

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  Fig. 8.. Schematic illustration of the proposed gate coupling model. Two gating elements, namely the width of the filter entrance and the hydration level of the central cavity, are coupled to modulate ion permeation. This coupling is mediated through interactions between the threonine at the filter entrance and the hydrophobic residue at the kink of the inner helix. This model applies to all examined channels, although the exact structural details depend on the channel type.