|
Fig. 1. I384 residue controls electromechanical coupling in the Shaker potassium channel.A Structure of the Shaker channel (PDB:7SIP). Voltage-sensing domains (VSD) and pore domains (PD) and S4–S5 helixes are labeled. B Location of I384 at the VSD–PD interface and its contacting residues. C, D The I384C mutant shows markedly slower deactivation compared to WT, with GV curves (N = 4) sharper and more closely aligned with QV (N = 5). Inset shows the tail current of I384C in black and WT in red returning to −120 mV. E, F WT displays typical ionic and gating currents, with GV and QV (n = 3 for both) separated. G–H I384L requires stronger depolarization for activation, with a shallower GV slope (N = 4) and altered gating current (N = 5) The inset highlights the coexistence of both gating and ionic current at the beginning of the pulse. I, J Fluorescence recordings from a TMR labeled I384L (A359C) suggest normal VSD movements (N = 3 for GV/QV/FV curves). Note there is no slow component in the fluorescence traces. K Single-channel recordings from I384L reveal unchanged conductance but increased flickering. L, M I384R abolishes ionic conduction in 12 external K+, leaving only gating currents, with the QV shifted strongly to negative voltages (N = 4 for I384R). N Gating current kinetics of I384R are faster than WT in the W434F background (N = 3 for both). All the data are shown as Mean ± SEM and N is the number biological replicates. For C, E, G, prepulse and returning pulse are both -120 mV and ∆V = 10 mV. For D, F, H, the data were fitted with a two-state model (details in method section) for easy visualization and calculation of change in ∆V1/2. Fitting results could be found in Supplementary Tables 1-2.
|
|
Fig. 2. Cryo-EM structure of uncoupled I384R channel, captured in a closed state.A Side view of I384R. The model is shown overlapping with the electron density, with the intracellular T1 domain omitted. Global resolution is 3.5 Å. B Intracellular view of I384R. Like previous solved structures, I384R adopts a domain-swapped arrangement. C Structure of the pore in I384R (green) compared to the open state (orange, PDB:7SIP) from the side view. A rotational movement in upper S6 helices (highlighted by arrows) positions two hydrophobic residues, I470 and V474, from the side directly into the permeation pathway. D Intracellular view of the pore in I384R (green) and WT (orange). In addition to the rotational movement, a translational movement of S6 helices towards the pore is observed in the closed state structure, further constraining the pore. E Radius profiles of the open pore from the WT channel. The inner cavity and bundle crossing are both open. The dashed vertical line indicates the approximate size of hydrated K+ ion. F Radius profile of pore in the uncoupled channel. The pore shows two constriction sites, one at I470 in the inner cavity and the other at V474 in the PVP motif. The Narrowest point of the pore is less than ~1 Å, indicated by the dashed red line. Clearly, the pore is captured in the closed state. G ANAP is a fluorescent unnatural amino acid sensitive to the hydrophobicity of its local environment. With the filter set used in this work, a more hydrophobic environment leads to an increase in fluorescence signal. H Representative ionic traces from I470ANAP. ANAP is incorporated at position 470 in a site-specific manner through amber stop codon suppression. I Fluorescence signal from I470ANAP. A transient signal could be seen among voltages where the channel opens (above −40 mV). A slightly slower signal is seen at the repolarizing pulse as well (highlighted by a red dashed line). At more negative voltages, however, no such signal could be resolved. It seems the observed fluorescence signal is associated with the opening and closing of the channel.
|
|
Fig. 3. A tripartite interaction pocket for electromechanical coupling and the structure of activated but not relaxed voltage sensors.A Intracellular view of the uncoupled structure (green) and coupled structure (orange) with their S4-S5 linkers highlighted. In the uncoupled structure, S4-S5 linkers undergo a translational movement towards the pore, forming a much tighter collar around the S6 helices. B, C In the coupled structure (orange), Y485 at the bottom of the S6 helix interacts with E395 and R394 in the C-terminus end of the S4-S5 linker from the adjacent subunit. I384 from the same subunit is lodged in a hydrophobic pocket formed by F484 and Y485. In the uncoupled structure (green), however, R384 jumps out of the hydrophobic pocket and forms a salt bridge with E395 in the adjacent unit, which repulses R394 away from facing the S6 helix, abolishing completely the interactions with Y485. D Side view of the VSD of the uncoupled channel and the WT channel. E Coulombic density for the VSD in I384R. All the side chains of the gating charges can be clearly resolved. F Comparison of the WT and I384R voltage sensors. In both cases, all gating charges (R362, R365, R368, and R371) have moved passed the hydrophobic plug around the I287 and F290 region. Both VSDs are in a fully activated state. G, H Hysteresis of the VSDs in WT and I384R (N = 4 for both). Holding at 0 mV for a prolonged time (>15 s) leads to a ~20 mV shift of the QV curve to the left in the WT channels. However, in I384R, no such shift was observed. It seems that at 0 mV, the VSDs in I384R do not enter the relaxed state. I Helical movements in S4 due to the S4-S5 linker. The tight conformation of the S4-S5 linker shifted the C terminus of the S4-S5 linker by 4.1 Å. This shift is transduced to the N-terminus end of the linker and the S4 helix as well, shifting them 1.7 Å and 1.4 Å, respectively. Data are shown as Mean ± SEM. N is the number of biological replicates.
|
|
Fig. 4. Noncanonical conformation of the selectivity filter and global decrease in protein volume in the closed state.A The SF captured in a closed state. The atomic model is overlaid with the density map. Clearly, there exist only two K+ in the SF. B Conductive SF captured in the open state. (PDB:7SIP). C Overlay of the SF in the closed (green) and open state (orange). In the closed state, K+ ions are seen bound at S2 and S4 positions. D Difference in SF between open and closed state structure. The carbonyl on the backbone of G446 flipped away from the permeation pathway, abolishing the S1 binding site on the top of the selectivity filter. A similar twist is seen at the bottom of the SF at the T442 position, resulting in a small displacement of the threonine side chain. E The channel is more expanded in the open state (orange) compared to the closed state (green). The expansion can be seen in the VSDs as well as the pore. F Cross-section area calculation utilizing CHARMM_GUI.
|
|
Fig. 5. Proposed activation mechanism and interactions with different functional states in Kv1 channels.The progression to activation is from left to right. In the deepest closed state of the channel, all the VSDs are down, and the SF likely resides in the noncanonical state (1). The closed pore is sealed closed by hydrophobic I470 and V474 (1, 2). Upon depolarization, VSDs transit to an active but not-relaxed state (3). The upward movement of the VSDs creates a pull on the S4-S5 linker, yet since not all VSDs are up, the energetic input is not enough to open the pore (4). In the last closed state before opening, or the pre-open state, all four VSDs move up (5), creating enough pull on the S4-S5 linkers (6). In the case of I384R, the channel is most likely stabilized in this pre-open state with newly introduced salt bridges. From the pre-open state, the last concerted movement happens, and the S6 helices undergo a roll and turn movement, opening up the permeation path and expanding the channel laterally (8). At the same time as the pore opens, the lateral movement of the S4-S5 linker drives the VSDs into a different state, the relaxed state (7).
|
