Identification of Intrahelical Bifurcated H-Bonds as a New Type of Gate in K+ Channels

Abstract

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K⁺ channels, Kcv_S and Kcv_NTS. These small viral encoded K⁺ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K⁺ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv_NTS and 40% in Kcv_S. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv_S. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

Figure 1

Chlorovirus encoded K⁺ channels. (a) Cartoon representations of a snapshot of Kcv_S taken from MD simulation at 80 ns of the production run. Left: Side view of the channel (two opposing monomer units). Right: view from bottom to top of the full tetrameric channel. Potassium ions are shown as red spheres, and F78 as explicit side chains in red. (b) Sequence alignment of three viral encoded K⁺ channels. The position of the transmembrane domains TMD1 and TMD2 as well as the pore helix are indicated by bars. The sequences are 86% identical; amino acid differences are highlighted in gray.

Figure 2

The difference in open probability of two similar K⁺ channels originates from a long closed time, which is absent in Kcv_NTS and present in Kcv_S. (a,b) Characteristic single channel fluctuations of Kcv_NTS and Kcv_S at different voltages in a planar lipid bilayer. The closed (C) and open (O) levels are indicated along the current traces. (c) Mean single channel i/V relations (±sd) of Kcv_NTS (open squares) and Kcv_S (filled squares) from 6 and 9 independent recordings, respectively. (d) Mean open probabilities (±sd) of the two channels from 6 and 9 independent recordings. Exemplary closed dwell times at +120 mV for Kcv_NTS (e) and Kcv_S (f). The data in e can be fitted with two exponentials while the data in f require a third exponential. The long-lived closed state in f is absent in e. The multiple kinetic variables of the two channels including the probabilities (P) for occupying the open state (O) and the closed states (C1–C3) (g) as well as mean lifetimes (in ms) of the open state (τ_o) and of the three closed states (τ_c1–τ_c3) (h) are presented in two-dimensional radar plots. The probabilities of occupancy and mean lifetimes were calculated from three independent 5 min recordings. The symbols in d, g and h correspond to those in c.

Figure 3

The Kcv_S long closed time is related to TMD2. (a) Chimeras were constructed by swapping transmembrane domains between Kcv_NTS (in blue) and Kcv_S (orange), for illustration purposes only. (b) Characteristic single channel fluctuations of chimeras Kcv_S/NTS and Kcv_NTS/S at ±120 mV in planar lipid bilayers. (c) Mean single channel i/V relations (±sd) of Kcv_NTS (open squares), Kcv_S (filled squares), Kcv_S/NTS (blue circles, n = 9) and Kcv_NTS/S (orange circles, n = 3). (d) Mean open probabilities (±sd) of wt channels and chimeras (n = 9 and n = 3 for Kcv_S/NTS and Kcv_NTS/S, respectively). Exemplary closed dwell times at +120 mV for Kcv_S/NTS (e) and Kcv_NTS/S (f). The data in e can be fitted with two exponentials while the data in f require a third exponential. Radar plots for probabilities (P) of the wt channels and the chimeras for occupying the open state (O) and the closed states (C1–C3) (g) and of mean lifetimes (in ms) of the open state (τ_o) and of the three closed states (τ_c1–τ_c3) (h). The probabilities of occupancy and mean lifetimes were calculated from three independent 5 min recordings. The symbols in d, g and h correspond to those in c.

Figure 4

The difference of Kcv_NTS and Kcv_S in position 77 is responsible for the absence or presence of a long closed time. Characteristic single channel fluctuations of mutant Kcv_S S77G (a) and Kcv_NTS G77S (b) at ±120 mV in planar lipid bilayers. (c) Mean single channel i/V relations (±sd) of Kcv_NTS (open squares), Kcv_S (filled squares), Kcv_NTS G77S (orange circles, n = 5) and Kcv_S S77G (blue circles, n = 3). (d) Mean open probabilities (±sd) of wt channels and mutants (n = 5 and n = 3 for Kcv_NTS G77S and Kcv_S S77G, respectively). Exemplary closed dwell times at +120 mV for Kcv_S S77G (e) and Kcv_NTS G77S (f). The data in e can be fitted with two exponentials while the data in f again require a third exponential. Radar plots for probabilities (P) of the wt channels and mutants for occupying the open state (O) and the closed states (C1–C3) (g) and for mean lifetimes (in ms) of the open state (τ_o) and of the three closed states (τ_c1–τ_c3) (h). The probabilities of occupancy and mean lifetimes were calculated from three independent 5 min recordings. The symbols in d, g and h correspond to those in c.

Figure 5

The aromatic AA F78 functions in combination with the upstream AA as a gate for the long closed time. Characteristic single channel fluctuations of mutant Kcv_S F78A (a) and Kcv_NTS G77S F78A (b) at ±120 mV in planar lipid bilayers. (c) Mean single channel i/V relations (±sd) of Kcv_NTS (open squares), Kcv_S (filled squares), Kcv_S F78A (orange circles, n = 4) and Kcv_NTS G77S F78A (blue circles, n = 7). (d) Mean open probabilities (±sd) of wt channels and mutants (n = 4 and n = 7 for Kcv_S F78A and Kcv_NTS G77S F78A, respectively). Exemplary closed dwell times at +120 mV for Kcv_S F78A (e) and Kcv_NTS G77S F78A (f). The data in e and f can now be fitted with two exponentials. Radar plots for probabilities (P) of the wt channels and mutants for occupying the open state (O) and the closed states (C1–C3) (g) and for mean lifetimes (in ms) of the open state (τ_o) and of the three closed states (τ_c1–τ_c3) (h). The mean lifetimes were calculated from three independent 5 min recordings. The symbols in d, g and h correspond to those in c.

Figure 6

The AA S77 is responsible for the gate with the long closed time. (a) Characteristic single channel fluctuations at +120 mV of Kcv_S and Kcv_NTS and mutants in which the position 77 in both channels was exchanged with an AA with a different flavor. (b) Mean open probabilities (±sd) of wt channels and mutants. The number of independent measurements is shown in brackets. Data in b correspond to the constructs on the left in a. Only AAs Ser and Thr in position 77 generate a low open probability with a long lasting closed time.

Figure 7

Dynamics of TMD2 in Kcv_S/Kcv_NTS and mutants. (a) Distribution of distance-angle pairs characterizing π-stack geometries in Kcv_NTS (top row) and Kcv_S (bottom row) averaged over all F78 pairs in opposite (left column) and neighboring (right column) monomers. The geometric criteria are shown Figure S5. Numbers in the left panel denote different monomers. (b) Analogous data for Kcv_S S77G and Kcv_NTS G77S mutants, demonstrating the transfer of π-stack characteristics upon mutating the key residues. (c) Time series of the distances between the carbonyl oxygen of I73 and the hydroxyl hydrogen of S77 for all four monomers. (d) Schematic representation of a hydrogen bond formed between the amide oxygen of I73 and the hydroxyl hydrogen of S77 within monomer 3 of Kcv_S supplemented by orientation of F78 (snapshot taken at 80 ns). (e) Snapshots of F78 (red) in tetramer of Kcv_NTS (top) and Kcv_S (bottom) representing a wider and a narrow translocation pathway corresponding to statistics shown in a.

Figure 8

The two channels Kcv_S and Kcv_NTS have a different orientation of the C-terminus. Characteristic single channel fluctuations at +120 mV of Kcv_S W82C (a) and Kcv_NTS W82C (b) in absence (−DTT) and presence of 5 mM DTT (+DTT) in bath solution. The prevailing open (O) and closed (C) levels are indicated along the traces.