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. 2018 Jul 17;115(29):E6751-E6759.
doi: 10.1073/pnas.1719105115. Epub 2018 Jun 29.

S4-S5 linker movement during activation and inactivation in voltage-gated K+ channels

Tanja Kalstrup  1 , Rikard Blunck  2   3
Affiliations

S4-S5 linker movement during activation and inactivation in voltage-gated K+ channels

Tanja Kalstrup et al. Proc Natl Acad Sci U S A. .

Abstract

The S4-S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4-S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4-S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4-S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4-S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4-S5 linker and pore opening disappears.

Keywords: Anap; inactivation; unnatural amino acids; voltage-clamp fluorometry; voltage-gated potassium channels.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of KV channel structure. (A) Topology of a Shaker subunit with relevant positions highlighted. A cysteine was inserted at position A359 for TMR labeling (green), and Anap was inserted into the S4–S5 linker (red). (B) Bottom view of the KV1.2–2.1 crystal structure of the homotetrameric transmembrane segment (3). At the bottom the S4–S5 linker sequence is shown, which was scanned for Anap incorporation. Anap was successfully incorporated into positions marked in red. The asterisks mark positions used to characterize the S4–S5 linker movement.
Fig. 2.
Fig. 2.
Fluorescence profile of conducting channels. (A) VCF recordings of (Upper) ionic currents, (Middle) Anap, and (Lower) TMR (green) fluorescence changes obtained from oocytes expressing each of the S4–S5 linker mutants. The arrow next to each fluorescence signal represents 1% ΔF/F. Anap FV, TMR FV, and GV curves of (B) L382Anap, (C) R387Anap, (D) K390Anap, and (E) A391Anap channels. Each dataset is fitted to a Boltzmann distribution (Methods). Error bars indicate mean ± SD. (F and G) Current and fluorescence output upon depolarization from −90 to 20 mV before and 30 min after external application of 40 mM barium in (F) R387Anap and (G) K390Anap channels. (Inset) Anap FV obtained before and after block. (H) Anap fluorescence (black) and ionic currents (red) of A391Anap channels upon prolonged 1-s depolarization from −90 mV to (Left) 50 and (Right) 100 mV. In gray, ionic current and Anap fluorescence after application of 5 mM external 4-AP for the 50-mV pulse is shown.
Fig. 3.
Fig. 3.
Fluorescence profile of nonconducting channels. (A) VCF recordings of (Upper) gating currents, (Middle) Anap, and (Lower) TMR (green) fluorescence changes obtained from oocytes expressing each of the S4–S5 linker mutants with the W434F mutation. Anap FV, TMR FV, and QV curves of (B) L382Anap-W434F, (C) R387Anap-W434F, (D) K390Anap-W434F, and (E) A391Anap-W434F channels. Except for Anap FV in D and E, each dataset is fitted to a Boltzmann distribution (Methods). Error bars indicate mean ± SD. (F) Anap fluorescence of K390Anap-W434F upon depolarization from −90 mV before (black) and after (gray) external application of 5 mM 4-AP. (G) Same as F but for A391Anap-W434F channels. (H and I) Anap FV before (filled symbols) and after (open symbols) 4-AP from (H) K390Anap-W434F and (I) A391Anap-W434F channels. The FV obtained after 4-AP was fitted to a Boltzmann distribution.
Fig. 4.
Fig. 4.
Kinetical analysis of fluorescence and gating current time course. (A–D) Time constants obtained from single or double exponential fits of TMR fluorescence (green), Anap fluorescence (red), and gating currents (black) for each of the S4–S5 linker mutants during a test pulse. (E) Overlapping TMR (green), Anap (red), and charge displacement (black) for an oocyte expressing L382Anap-W434F. Arrow highlights kinetic correlation between the fast component of Anap fluorescence and charge displacement. (F) Structural view from the KV1.2–2.1 crystal structure (3) showing the four S4–S5 linker positions in red and the distance from L382 to V234 in green in S1.
Fig. 5.
Fig. 5.
Comparison of onset of TMR and Anap fluorescence signal. (A–F) Overlap of TMR (green) and Anap (red) fluorescence upon depolarizations from −90 mV. Arrows in A and D highlight the absence of a delayed onset, whereas arrows in B, C, E, and F highlight the presence of a delayed onset for Anap compared with TMR. In the boxes a vertical zoom of the curves is shown to better visualize the delay. The depolarizations were chosen with respect to the respective V1/2 values. In the zoomed display, the data were filtered by moving average to remove white noise. (G) Voltage dependence of the delay for all three mutants. Solid symbols indicate -W434F mutants; open symbols indicate conducting. The fit relates to the W434F data, which were obtained with better signal-to-noise ratio.
Fig. 6.
Fig. 6.
Separation of the final gating transition using F290A. (A and B) (Upper) Gating currents and (Lower) Anap fluorescence changes. The arrows highlight endogenous leak currents during high depolarizations. Anap FV and QV for (C) F290A-R387Anap-W434F and (D) F290A-K390Anap-W434F plotted together with the GV obtained from the respective conducting mutants. (E) QVon (gray) and QVoff (black) obtained from the gating currents in A and B, respectively. For comparison with QVon, QVoff has been normalized to the amplitude of the saddle point. (F) QV, Anap FV, and GV for Shaker-H486Anap-W434F. (G) C-type inactivation of H486Anap. Current and fluorescence change elicited upon a 1-s depolarization from −90 to +100 mV to induce C-type inactivation in H486Anap channels.
Fig. 7.
Fig. 7.
Stylized model for cytosolic gating machinery. (Upper) The spots indicate the positions from which we obtained fluorescence data. (Lower) Voltage sensors switch between resting (R), activated (A), and relaxed (X) state, indicated by orange letters; the pore assumes the closed (C), open (O), and inactivated (I) state, indicated by blue letters. The channel undergoes transitions A–D. Here the spots indicate at which position the respective transition has been detected. Voltage sensor activation was detected with a temporal delay toward the C terminus of the S4–S5 linker, which is reflected in the spot color for this transition (blue before orange). The model is shown for (Left) wild type and (Right) the W434F mutant.
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