Lacroix JJ , Pless SA , Maragliano L , Campos FV , Galpin JD , Ahern CA , Roux B , Bezanilla F .

56858 Canadian Institutes of Health Research , GM030376 NIGMS NIH HHS , GM062342 NIGMS NIH HHS , R01 GM062342 NIGMS NIH HHS , R01 GM106569 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS

	Figure 1. Effect of I241 mutations on Shaker channel gating. (A) Zoomed view of the refined structure of the Kv1.2 voltage sensor (PDB 3LUT; Chen et al., 2010) showing the S1 (white), S2 (yellow), S3 (red), and S4 (blue) helices, the four S4 Arg R1–R4 and the residues I241. (B) Ionic current traces for WT Shaker (top) and I241Y mutant (bottom) recorded using the indicated protocol. (C) G-V curves of Shaker channels as a function of the residue present at position 241. The curves are colored gradually from blue to red relative to their position on the voltage axis from negative to positive potentials. (D) Sequence alignments of the S1 segment (T-Coffee) showing the amino acid at the position homologue to I241 (boxed) in various voltage sensors containing proteins (Shaker, GI: 13432103; Kv1.2, GI: 4826782; Kv2.1, GI: 4826784; Kv3.1, GI: 76825377; Kv4.1, GI: 27436981; Kv7.1, GI: 32479527; Shaw, GI: 220901931; KvAP, GI: 38605092; Nav1.4 domain I, GI: 292495096 and Ci-VSP, GI: 76253898).
	Figure 2. The I241 mutations alter VSD movement. The figure shows the gating currents traces for WT Shaker (A), the I241T (B), and I241F (C) mutants recorded using the indicated protocols. (D) Q-V curves for the WT and I241 mutants. (E and F) Correlation plots between the mid-points of the G-V (V1/2G-V) and Q-V (V1/2Q-V) (E) and between V1/2G-V and the difference V1/2G-V – V1/2Q-V (F). The line represents a linear fit to the data (R2 = 0.97). The coloring method in D–F is identical to Fig. 1 C.
	Figure 3. I241W transiently immobilizes S4. (A) Family of gating currents recording for the I241W mutant. (B) Q-V plots for the WT and I241W channels. (C) Family of fluorescence recordings of a tetramethylrhodamine-maleimide fluorophore attached to M356C in the I241W mutant. (D) F-V curve of the labeled I241W/M356C mutant obtained from an isochronal plot of the fluorescence intensities at the end of 300-ms test pulses.
	Figure 4. Hypothetical mechanisms for the I241W phenotype. (A) Hypothetical energy diagram of the VSD transitions showing the resting (R), intermediate (I), and active (A) states at a voltage of 0 mV. The dotted lines indicate possible effects of the I241W mutation: destabilization of the active state (left), increase of the energy barrier between the intermediate and active states (middle), and stabilization of the intermediate state (right). (B) Activation ionic currents recordings for I241W channels. (C) Deactivation ionic currents recordings for I241W channels. (D) τ–V plots for WT and I241W channels during activation. (E) τ–V plots for WT and I241W channels during activation.
	Figure 5. The I241W phenotype depends on the S4 Arg. The figure shows the Q-V curves measured during an activation pulse protocol for the mutants R1K (A, black squares), R2K (B, black squares), R3K (C, black squares), R4K (D, black squares), and the double mutants I241W/R1K (A, open circles), I241W/R2K (B, open circles), I241W/R3K (C, open circles), and I241W/R4K (D, open circles). In each graph, the dotted blue line represents the Q-V curve for the I241W mutant and the red line represents the fit to a sequential double-Boltzmann equation. (E) Histogram showing the s values (s = e0(V2z2 −V1z1)/kT) (see Table 1). A positive value indicates the presence of a stabilized intermediate state.
	Figure 6. Conservation of the I241W phenotype during deactivation. (A) Deactivation gating current traces recorded in an oocyte expressing I241W channels with the indicated protocol at 18°C. (B) Deactivation gating currents traces recorded in the same cell at 28°C. Traces at the post-pulses in A and B are shown expanded for clarity. (C) τ-V curves of gating currents for the I241W mutant recorded at 18°C (black symbols) or 28°C (open symbols) during activation (circles) or deactivation (squares). (D) Q-V curves for the I241W mutant determined at 28°C during activation (black squares) or deactivation (open circles). (E) Q-V curves for the I241W/R2K mutant determined at 28°C during activation (black squares) or deactivation (open circles). (F) Q-V curves for the I241W/R3K mutant determined at 28°C during activation (black squares) or deactivation (open circles).
	Figure 7. The F244W mutation mimics the I241W mutation. (A) Ionic current traces recorded from the F244W mutant using the indicated activation protocol. (B) Gating current traces recorded from the F244W mutant using the indicated activation protocol. (C) Q-V (black squares), G-V (open squares), and τ-V of gating current (black spheres) plots for the F244W mutant determined from activation protocols. (D) Q-V curves for the double mutants F244W/R2K (black squares) and F244W/R3K (open circles) determined from activation protocols. The dotted blue line represents the Q-V curve of the F244W mutant depicted in C. The red lines represent the fit to a sequential double-Boltzmann equation. (E) Deactivation gating currents measured for the F244W mutant at 28°C. (F) Q-V curves determined at 28°C during activation (black squares) and deactivation (open circles) for the F244W mutant.
	Figure 8. Investigations of the I241W and F244W mutants with nonnatural amino acids. (A, left) Representative current recordings of Shaker channel bearing the natural Trp or the unnatural F4-Trp side chain at position 241. 50-ms depolarizing pulses were used in 5-mV increments from −20 mV to +100 mV with a holding potential of −80 mV. (Right) G-V curves for WT I241 (black circles, V1/2 = −22.3 ± 0.6 mV; Z = 2.9 ± 0.3, n = 5), I241Trp (black squares, V1/2 = 41.9 ± 0.5 mV; Z = 1.9 ± 0.1; n = 5) and I241F4-Trp (open diamonds, V1/2 = 51.9 ± 1.3 mV; Z = 1.8 ± 0.2; n = 5). (B, left) Representative current recordings of Shaker channel bearing the Ind side chains at position 241. 50-ms depolarizing pulses were used in 5-mV increments from −60 mV to +60 mV with a holding potential of −80 mV. (Right) G-V curves for WT I241 (black circles, V1/2 = −22.3 ± 0.6 mV; Z = 2.9 ± 0.3; n = 5) and the I241-Ind mutant (open triangles, V1/2 = 0.6 ± 2.9 mV; Z = 2.5 ± 0.3; n = 6). (C) G-V curves of WT Shaker (black circles), the F244Trp mutant (black squares), the F244F4-Trp mutant (open diamonds), and the F244-Ind mutant (open triangles).
	Figure 9. Simulation of the VSD trajectory using the string method. (A) The figure shows selected snapshots of VSD conformations along the converged path from the string method. The numbers indicate the positions from the initial linear path between the up (image #1) and down (image #32) states. The coloring method for S1–S4 is the same as in Fig. 1 A. The side chains of F233 (F290 in Shaker), I177 (I241 in Shaker) E0-2, D3, and R1–R4 are shown in all snapshots in licorice representation. (B) Vertical Z-distance of the S4 arginines relative to the center-of-mass of F233 side chain along the optimized path. The solid and dashed lines correspond to the distance relative to the R1–4 Cζ and R1–4 Cα, respectively. (C) Transient salt bridges analysis along the optimized path; according to the color code, a color corresponding to a particular arginine/lysine is drawn at an image if in the corresponding configuration there is a salt bridge between the basic and the acidic residue. The index of the images along the path is reported on the x-axis, and the acidic amino acids making interactions with the S4 arginines are reported on the y-axis. The horizontal dashed line schematically separates the extracellular region (above) from the intracellular (below).
	Figure 10. A possible mechanism for the I241W and F244W phenotypes. (A) Graph showing the distances between I241 (Cβ) and R1–R4 (Cζ) along the pathway obtained by the string method. The mutations I241W (B) and I241W/R2K (C) were independently introduced in silico in the conformation having the minimal I241-R2 distance (image #25) and equilibrated for 32 ns. Only the side chains were allowed to move during the two equilibrations. (D) The graph shows the vdW (top) and electrostatic (bottom) interaction energies between I241W and R2 (black) or between I241W and R2K (red) during the two simulations. (E) The mutation F244W was introduced in silico in image #22 giving the minimal F244-R2 (Cβ-Cζ) distance and equilibrated for 20 ns. The cartoon shows the side chains of R2 and F244W after equilibration. (F) The graph shows the vdW (top) and electrostatic (bottom) interaction energies between F244W and R2 during the simulation.
	Figure 11. Simulations with the 3-state model. (A) Q-V curves computed for several values of K1 and K2, as follows (a): K1 = 0.0001, K2 = 166.7, s = 14.8; (b): K1 = 0.0004, K2 = 27.8, s = 11.2; (c): K1 = 0.0022, K2 = 4.63, s = 7.67; (d): K1 = 0.013, K2 = 0.77, s = 4.09; (e): K1 = 0.078, K2 = 0.129, s = 0.5. The next three curves are (f): K1 = 0.467, K2 = 0.021, s = −3.08; (g): K1 = 2.8, K2 = 0.0036, s = −6.66; and (h): K1 = 16.8, K2 = 0.0006, s = −10.25. Curves e–h can be well fit by a single Boltzmann given apparent valences of 2.36, 3.54, 3.91, and 3.99, respectively. (B) First derivative of the Q-V curves shown in part A. (C) Q-V and weighted time constant versus V curves computed with the 3-state model with the following parameters: z1f = 0.5, z1b = 1.5, z2f = 1.2, z2b = 0.8, α10 = 0.1, β10 = 0.001, α20 = 0.001, β20 = 0.1.

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed , Xenbase
Ahern, Focused electric field across the voltage sensor of potassium channels. 2005, Pubmed , Xenbase
Ahern, Electrostatic contributions of aromatic residues in the local anesthetic receptor of voltage-gated sodium channels. 2008, Pubmed , Xenbase
Ahern, A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels. 2006, Pubmed
Armstrong, Currents related to movement of the gating particles of the sodium channels. 1973, Pubmed
Asamoah, A fluorometric approach to local electric field measurements in a voltage-gated ion channel. 2003, Pubmed
Ben-Chaim, Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor. 2006, Pubmed , Xenbase
Campos, Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel. 2007, Pubmed , Xenbase
Cha, Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. 1997, Pubmed , Xenbase
Chen, Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement. 2010, Pubmed
DeCaen, Sequential formation of ion pairs during activation of a sodium channel voltage sensor. 2009, Pubmed
DeCaen, Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation. 2008, Pubmed
DeCaen, Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor. 2011, Pubmed
Delemotte, Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations. 2011, Pubmed
Dougherty, Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. 1996, Pubmed
E, Finite temperature string method for the study of rare events. 2005, Pubmed
Gan, Atomistic view of the conformational activation of Src kinase using the string method with swarms-of-trajectories. 2009, Pubmed
Haddad, Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain. 2011, Pubmed , Xenbase
Henrion, Tracking a complete voltage-sensor cycle with metal-ion bridges. 2012, Pubmed , Xenbase
HODGKIN, A quantitative description of membrane current and its application to conduction and excitation in nerve. 1952, Pubmed
Islas, Electrostatics and the gating pore of Shaker potassium channels. 2001, Pubmed , Xenbase
Jacobson, Calcium-activated and voltage-gated potassium channels of the pancreatic islet impart distinct and complementary roles during secretagogue induced electrical responses. 2010, Pubmed
Jensen, Mechanism of voltage gating in potassium channels. 2012, Pubmed
Khalili-Araghi, Calculation of the gating charge for the Kv1.2 voltage-activated potassium channel. 2010, Pubmed
Kirmizialtin, How conformational dynamics of DNA polymerase select correct substrates: experiments and simulations. 2012, Pubmed
Lacroix, Control of a final gating charge transition by a hydrophobic residue in the S2 segment of a K+ channel voltage sensor. 2011, Pubmed , Xenbase
Lacroix, Properties of deactivation gating currents in Shaker channels. 2011, Pubmed
Lin, R1 in the Shaker S4 occupies the gating charge transfer center in the resting state. 2011, Pubmed , Xenbase
Lin, Transfer of ion binding site from ether-a-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening. 2010, Pubmed , Xenbase
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Long, Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. 2007, Pubmed
Mackerell, Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. 2004, Pubmed
Mannuzzu, Direct physical measure of conformational rearrangement underlying potassium channel gating. 1996, Pubmed , Xenbase
Maragliano, String method in collective variables: minimum free energy paths and isocommittor surfaces. 2006, Pubmed
Miller, Solvent coarse-graining and the string method applied to the hydrophobic collapse of a hydrated chain. 2007, Pubmed
Murata, Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. 2005, Pubmed , Xenbase
Muroi, Molecular determinants of coupling between the domain III voltage sensor and pore of a sodium channel. 2010, Pubmed
Ovchinnikov, Free energy of conformational transition paths in biomolecules: the string method and its application to myosin VI. 2011, Pubmed
Pan, Finding transition pathways using the string method with swarms of trajectories. 2008, Pubmed
Pathak, Closing in on the resting state of the Shaker K(+) channel. 2007, Pubmed
Payandeh, The crystal structure of a voltage-gated sodium channel. 2011, Pubmed
Perozo, Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. 1993, Pubmed
Petheo, Molecular and functional characterization of Hv1 proton channel in human granulocytes. 2010, Pubmed
Phillips, Scalable molecular dynamics with NAMD. 2005, Pubmed
Pless, Contributions of counter-charge in a potassium channel voltage-sensor domain. 2011, Pubmed , Xenbase
Pless, Molecular basis for class Ib anti-arrhythmic inhibition of cardiac sodium channels. 2011, Pubmed
Pless, A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists. 2011, Pubmed , Xenbase
Posson, Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer. 2005, Pubmed , Xenbase
Posson, Extent of voltage sensor movement during gating of shaker K+ channels. 2008, Pubmed , Xenbase
Ramsey, A voltage-gated proton-selective channel lacking the pore domain. 2006, Pubmed
Ruta, Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. 2005, Pubmed
Sasaki, A voltage sensor-domain protein is a voltage-gated proton channel. 2006, Pubmed
Seoh, Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. 1996, Pubmed , Xenbase
Smith-Maxwell, Role of the S4 in cooperativity of voltage-dependent potassium channel activation. 1998, Pubmed , Xenbase
Starace, A proton pore in a potassium channel voltage sensor reveals a focused electric field. 2004, Pubmed
Starace, Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel. 2001, Pubmed , Xenbase
Tao, A gating charge transfer center in voltage sensors. 2010, Pubmed , Xenbase
Vargas, An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations. 2012, Pubmed
Vargas, In search of a consensus model of the resting state of a voltage-sensing domain. 2011, Pubmed
Villalba-Galea, S4-based voltage sensors have three major conformations. 2008, Pubmed
Zhong, From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. 1998, Pubmed
Zhu, Pore opening and closing of a pentameric ligand-gated ion channel. 2010, Pubmed