Zhao J , Blunck R .

MOP-136894 CIHR, MOP-102689 CIHR

	Figure 1. Expression of Shaker-iVSD lacking pore domains is sufficient to reconstitute voltage-dependent channel activity.(a) Top: topology of Shaker K+ channel. Amino acid positions relevant to the present study are highlighted in yellow. Middle: Shaker channels constructs (Shaker-W434F and Shaker-iVSD) used in the present study. Thiol-reactive fluorophores are attached to a cysteine at position A359C in the S3–S4 linker. The mutation W434F in the pore region produces an instantly C-type inactivated Shaker channel. Shaker-iVSD is a deletion mutant truncated after the S4 helix. Bottom: Sequence of the C-terminus of Shaker-iVSD (b) Gating currents recorded from oocytes expressing Shaker-iVSD (top) and Shaker-W434F (bottom) using cut-open oocyte voltage clamp. Leak, background, and capacitive currents were subtracted from the current traces using a P/4 protocol. Inset shows the current step protocol. Gating currents were elicited by test pulses from −100 mV to +120 mV at a holding potential of −90 mV. Zero level was indicated by the red dashed line. (c and d) Representative ionic currents recorded by cut-open oocyte voltage-clamp from oocytes expressing Shaker-iVSD and Shaker-W434F. At depolarized (0 mV, top) or hyperpolarized (−90 mV, bottom) holding potentials, currents were elicited by voltage pluses at a range between −180 and +140 mV in 10 mV increments without leak substraction. The interval between test pulses was 5 s to allow complete recovery of the channels. An inset on top of current traces shows the corresponding voltage protocols. Zero level was indicated by the red dashed line. NMDG+-based solutions were used as the external and internal solutions, pHin/pHout 7.35/7.35. (e) Representative ionic currents recorded from HEK293 cells expressing Shaker-iVSD using the whole-cell configuration of the patch-clamp technique. Cells were depolarized for 500 ms to potentials ranging from –140 mV to +100 mV (at a holding potential of 0 mV, top), or from −140 mV to +80 mV (at a holding potential of −90 mV, bottom) in 10 mV increments. An inset on top of current traces shows the corresponding voltage protocols. Zero level was indicated by the red dashed line. To compare with the ionic current recorded from oocytes as shown in c, NMDG+-based solutions were also used as the extracellular and intracellular solutions, pHin/pHout 7.35/7.35. (f) I-V relations of currents recorded from Shaker-iVSD-injected oocytes at holding potentials of 0 mV (black triangle, n = 14) or −90 mV (red triangle, n = 13), and H2O-injected oocytes at holding potentials of 0 mV (blue triangle, n = 5) using the cut-open oocyte voltage clamp technique (see protocol in the upper inset of c). The mean steady-state currents during the last 50 ms of the command pulses were plotted versus voltage. (g) I-V relations of ionic currents recorded from HEK293 cells transfected with Shaker-iVSD at holding potentials of 0 mV (black triangle, n = 8) or −90 mV (red triangle, n = 4) using the whole-cell patch clamp technique (see protocol in the upper inset of e). The mean steady-state currents during the last 50 ms of the command pulses were plotted versus voltage.DOI: http://dx.doi.org/10.7554/eLife.18130.003
	Figure 2. Selectivity of Shaker-iVSD-induced current in high buffer (HB) solutions or NMDG+-based solutions.(a)Top. Normalized IV relationships of Shaker-iVSD induced currents under different external pH conditions with HB solutions. Tail currents were evoked by 250 ms voltage pulses ranging from −180 to +120 mV following a 200 ms hyperpolarization prepulse to −120 mV using cut-open oocyte technique (see inset for protocol). External HB solution contained (in mM) 70 NMDG+, and either 180 acetic acid (for pH 4.5), 180 HEPES (for pH 7.5), or 136 CHES with 44 D-sorbitol (for pH 9.5). The composition of the HB internal solution was the same as the HB internal solution at pH 7.5. Bottom. Same normalized IV relationships, but zoomed in to emphasize the changes of Vrev. At pHin/pHout 7.5/7.5, Vrev = −0.14 ± 0.18 mV (black triangle, n = 56); at pHin/pHout 7.5/4.5, Vrev = 12.5.2 ± 1.6 mV (blue triangle, n = 5); at pHin/pHout 7.5/9.5, Vrev = −5.4 ± 1.76 mV (red triangle, n = 5). (b) Top. Normalized IV relations of currents with NMDG+in/NMDG+out 115 mM/10 mM or 115 mM/115 mM, at pHin/pHout 7.35/7.35. NMDG+-based external and internal solutions contained 115 mM NMDG-MeSO3 were the same as in Figure 1b–d. To test the permeability of Shaker-iVSD-induced currents to NMDG+ ions, 115 mM NMDG-MeSO3 in the external solution was replaced by 10 mM NMDG-MeSO3 and 210 mM D-sorbitol, at pH 7.35. Currents were elicited by the voltage pulse protocol shown in the inset. Bottom. Same normalized IV relationships, but zoomed in to emphasize the changes of Vrev. At NMDG+in/NMDG+out 115 mM/10 mM, Vrev = −62.6 ± 2.5 nmV (red triangle, n = 4); at NMDG+in/NMDG+out 115 mM/115 mM, Vrev = −0.8 ± 1.1 (black triangle, n = 4). (c) Top. Normalized IV relationships of currents recorded from HEK293 cells transfected with Shaker-iVSD cDNA with HB solutions. Tail currents were elicited from a holding potential of 0 mV by 250 ms voltage pulses ranging from –140 to +100 mV in 10 mV increments, following a 200 ms hyperpolarization prepulse to −120 mV using whole-cell patch clamp technique (see inset for protocol). The intracellular and extracellular HB solutions contained (in mM) 10 NMDG+, and either 24 HEPES (for PH 7.5), 56 MES (for pH5.5), or 35 acetic acid (for pH4.5). Osmolarity was adjusted to 300~320 mOsm by D-sorbitol. Bottom. Same normalized IV relationships, but zoomed in to emphasize the changes of Vrev. Vrev values were 0.5 ± 0.2 mV at pHin/pHout 7.5/7.5 (black triangle, n = 9), −7.0 ± 1.1 mV at pHin/pHout 5.5/7.5 (red triangle, n = 6), and −34.9 ± 1.8 mV at pHin/pHout 4.5/7.5 (blue triangle, n = 10). (d–e) Comparison of conductance of Shaker-iVSD for external NMDG+ (black triangles, n = 12), K+ (f, red triangles, n = 7) and Na+ (g, blue triangles, n = 7) using cut-open oocyte technique. The NMDG+-based external solution and internal solutions were the same as in Figure 1b–d at pHin/pHout 7.35/7.35. Na+-based or K+-based external solutions were the same as the NMDG+-based external solution except that 115 mM NMDG- MeSO3 was replaced by the same concentration of NaOH- MeSO3 or KOH- MeSO3. Currents were elicited by the voltage pulse protocol shown in the inset of Figure 1c, and currents were normalized relative to the maximum NMDG+ inward currents and plotted versus voltage. (f) Replacing MeSO3 with chloride (green triangle, n = 7) in the external NMDG+-based solution did not exhibit significantly difference from the currents recorded in external NMDG-MeSO3 solution, indicating that it is likely not carried by anions.DOI: http://dx.doi.org/10.7554/eLife.18130.004
	Figure 3. Inhibition of Shaker-iVSD-induced ionic currents by external ZnCl2.(a) Ionic currents and normalized fluorescence traces recorded from oocytes expressing Shaker-iVSD before and after the application of 2~20 µM ZnCl2 using cut-open technique (see inset for protocol). Zero level is indicated by the grey dashed line. To illustrate the effects of ZnCl2 on kinetics, the same ionic currents were normalized to the value at the end of the 250 ms pulse (see lower inset). (b) Fluorescence traces to the recordings in a obtained from fluorescent labeling at position A359C atop S4. (c) IV-relations of Shaker-iVSD at increasing concentrations of ZnCl2. (for protocol see a). Currents were normalized to the maximal current amplitude in the absence of Zn2+. (d) Normalized FV relations in the absence (black) and presence of external 2 μM (red) or 10 μM (green) ZnCl2. Fluorescence curves were generated using the protocol shown as inset. Smooth curves are fits to a Boltzmann function yielding the following V1/2 and dV values: −88.2 ± 1.2 and 32.2 ± 1.7 mV for Shaker-iVSD control (n = 6); −89.6 ± 2.3 mV and 32.8 ± 2.6 mV for 2 μM ZnCl2(n = 6); −98.4 ± 2.9 mV and 37.5 ± 2.0 mV for 10 μM ZnCl2(n = 6). Each curve was normalized to its own maximal relative change dF/F. (e) Effect of Zn2+ on relative fluorescence change (dF/F, grey) and ionic current. The relative fluorescence change remains constant even during bleaching (Blunck et al., 2004), assuming not too high background fluorescence. A reduction of the relative fluorescence change is thus correlated with immobilization of the voltage sensors. The current values are the maximal current amplitudes of c normalized to the value in the absence of Zn2+. (f) Position of H140 (S2) and H193 (S3-S4 linker; numbering according to human Hv1) in the crystal structure of Hv1 (PDB: 3WKV) in the entry to the gating pore. Missing residues in the crystal structure were modeled using Modeller (Sali and Blundell, 1993). (g) Ionic current traces recorded from oocytes expressing Shaker-iVSD D277H in the absence (black) and presence (red) 0.1 µM ZnCl2 using cut-open technique (see inset in a for protocol). (h) Normalized FV relations in the absence (black triangle, n = 5) and presence (red triangle, n = 5) of 0.1 µM ZnCl2. Fluorescence signals were generated using the protocol shown as inset in d. Smooth curves are Boltzmann fits to the data with the following V1/2 and dV values: −99.0 ± 2.5 mV and 25.9 ± 1.3 for control; −110.4 ± 0.2 mV and 25.7 ± 1.9 mV for ZnCl2. (i) Dose-response curves of inhibition by external ZnCl2 for Shaker-iVSD and Shaker-iVSD D277H ionic currents. Data points correspond to normalized amplitudes of ionic currents elicited by step hyperpolarization to −150 mV (As shown in a), plotted as a function of ZnCl2 concentration, and fitted with the Hill equation. The IC50 value of ZnCl2 for the Shaker-iVSD D277H inward currents was 158.4 nM (maximum inhibition 79%, n = 5), which was significantly lower than the IC50 value obtained from Shaker-iVSD currents 6.04 µM (maximum inhibition 80%, n = 6).DOI: http://dx.doi.org/10.7554/eLife.18130.005Figure 3—figure supplement 1. Effect of Zn2+ on fluorescence (left) and gating currents (right).Fluorescence changes reduced significantly upon addition of Zn2+ and recover upon washout. Gating currents were inhibited upon addition of 5 µM Zn2+. At 5 µM Zn2+, tail currents at hyperpolarized potentials are still visible.DOI: http://dx.doi.org/10.7554/eLife.18130.006
	Figure 4. Conformational changes in Shaker-iVSD.Shaker-iVSD displays slow voltage-dependent fluorescence quenching in oocytes under voltage-clamp using cut-open oocyte technique. (a) Typical fluorescence signals are shown for Shaker-iVSD and Shaker-W434F for 250 ms pulses between −180 and +160 mV from holding potentials of −90 mV (left), 0 mV (center), and +90 mV (right). The inset shows the corresponding clamp protocol. (b) Scaled and overlaid representative ionic current (black) and fluorescence traces (red) in response to a voltage step from 0 mV to −150,–130, −110 and −90 mV for Shaker-iVSD (see inset for protocol). Note that the fluorescence signal has been inverted for a direct kinetic comparison with the current traces. (c) The activation kinetics of the Shaker-iVSD ionic currents (as shown on the top of Figure 1c) and fluorescence traces during channel activation (as shown in a) were fitted by two exponential components, Ifast and Islow for ionic currents (n = 7), Ffast and Fslow for fluorescence traces (n = 7). The fitted time constants were plotted as a function of test potential. (d) FV relationships of Shaker-iVSD and Shaker-W434F at holding potential of −90 mV calculated by normalizing the mean ΔF values during the last 50 ms of the command pulse to the maximum fluorescence change, and plotted against voltage. Smooth curves were fits to a Boltzmann function yielding the following V1/2 and dV values: −38.8 ± 3.4 and 49.3 ± 2.3 mV for Shaker-iVSD (n = 12), −49.2 ± 1.3 and 8.6 ± 0.4 mV for Shaker-W434F (n = 12).DOI: http://dx.doi.org/10.7554/eLife.18130.007
	Figure 5. Mode shift in Shaker-iVSD.(a) FV relationships at 7 different holding potentials of Shaker-iVSD (left) and Shaker-W434F (right). Smooth curves were fits to a Boltzmann function yielding the following V1/2 and dV values: for Shaker-iVSD, −38.8 ± 3.4 and 49.3 ± 2.3 mV at HP −90 mV (n = 12), −58.5 ± 2.5 and 57.4 ± 2.3 mV at HP −60 mV (n = 24), −77.0 ± 2.7 and 44.9 ± 1.3 mV at HP −30 mV (n = 20), −88.7 ± 1.4 and 33.6 ± 1.5 mV at HP 0 mV (n = 24), −98.2 ± 2.4 and 32.0¸± 2.0 mV at HP + 30 mV (n = 9), −107.1 ± 3.7 and 37.2 ± 2.0 mV at HP + 60 mV (n = 11), −133.7 ± 4.2 and 40.6 ± 1.9 mV at HP + 90 mV (n = 7); for Shaker-W434F, −49.2 ± 1.3 and 8.6 ± 0.4 mV at HP −90 mV (n = 12), −5.5 ± 3.0 and 14.2 ± 0.9 mV at HP −60 mV (n = 14), −75.4 ± 2.4 and 11.6 ± 1.1 mV at HP −30 mV (n = 14), −77.1 ± 2.0 and 11.2 ± 0.9 mV at HP 0 mV (n = 7), −77.7 ± 2.1 and 11.7 ± 0.8 mV at HP + 30 mV (n = 7), −78.9 ± 2.0 and 11.2 ± 0.8 mV at HP + 60 mV (n = 8), −80.3 ± 1.8 and 10.8 ± 0.5 mV at HP + 90 mV (n = 6). (b) V1/2 values from a plotted versus holding potential. (c–d) Normalized FV relations of Shaker-iVSD and Shaker-W434F from a holding potential of 0 mV to a pre-pulse of −90 mV for variable duration (5–5000 ms) followed by a series of test pulses of −180 to +160 mV in steps of 10 mV (see inset for protocol). For Shaker-iVSD, smooth curves are fits to a Boltzmann function with V1/2 values shown in e as a function of pre-pulse duration. For Shaker-W434F, the resulting distribution is a superposition of two Boltzmann curves representing the FV0 mV and the mode-shifted FV−90 mV. Smooth curves are fits of the data to a double Boltzmann function, and the fractional amplitudes of FV−90 mV component are shown in e as a function of pre-pulse duration. (e). Data points of Shaker-iVSD (red circle, right ordinate) were fitted by a single-exponential function with a time constant for entering the mode shift of 452 ms. Data points of Shaker-W434F (black circle, left ordinate) were fitted by a double-exponential function having time constants for entering the mode shift of 36 ms and 279 ms. (f) Comparision the time course for entering mode-shift with the development of the ionic current of Shaker-iVSD. The red data points are V1/2 values recorded from one oocyte plotted as a function of pre-pulse duration (5–1000 ms) (see protocol in c), and fitted by a single-exponential function (red curve). Ionic current recorded from the same oocyte (black) was scaled and overlaid with the red curve.DOI: http://dx.doi.org/10.7554/eLife.18130.008

Arrigoni, The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. 2013, Pubmed, Xenbase

Arrigoni, The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. 2013, Pubmed , Xenbase
Batulan, An intersubunit interaction between S4-S5 linker and S6 is responsible for the slow off-gating component in Shaker K+ channels. 2010, Pubmed
Bezanilla, The voltage-sensor structure in a voltage-gated channel. 2005, Pubmed
Blunck, Mechanism of electromechanical coupling in voltage-gated potassium channels. 2012, Pubmed
Blunck, Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells. 2004, Pubmed , Xenbase
Boland, Cysteines in the Shaker K+ channel are not essential for channel activity or zinc modulation. 1994, Pubmed , Xenbase
Bruening-Wright, Slow conformational changes of the voltage sensor during the mode shift in hyperpolarization-activated cyclic-nucleotide-gated channels. 2007, Pubmed , Xenbase
Catterall, Ion channel voltage sensors: structure, function, and pathophysiology. 2010, Pubmed
Cha, Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. 1997, Pubmed , Xenbase
Chakrapani, Structural dynamics of an isolated voltage-sensor domain in a lipid bilayer. 2008, Pubmed
Choi, Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. 1991, Pubmed
Cox, An SCN9A channelopathy causes congenital inability to experience pain. 2006, Pubmed
Cuello, Structural mechanism of C-type inactivation in K(+) channels. 2010, Pubmed
DeCoursey, Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. 2013, Pubmed
Faure, A limited 4 Å radial displacement of the S4-S5 linker is sufficient for internal gate closing in Kv channels. 2012, Pubmed
Haddad, Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain. 2011, Pubmed , Xenbase
Hille, Ionic channels in excitable membranes. Current problems and biophysical approaches. 1978, Pubmed
Jiang, X-ray structure of a voltage-dependent K+ channel. 2003, Pubmed
Kalstrup, Dynamics of internal pore opening in K(V) channels probed by a fluorescent unnatural amino acid. 2013, Pubmed , Xenbase
Kim, Evidence for functional diversity between the voltage-gated proton channel Hv1 and its closest related protein HVRP1. 2014, Pubmed
Kreusch, Crystal structure of the tetramerization domain of the Shaker potassium channel. 1998, Pubmed
Kuzmenkin, Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents. 2004, Pubmed
Labro, Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues. 2008, Pubmed
Labro, Molecular mechanism for depolarization-induced modulation of Kv channel closure. 2012, Pubmed , Xenbase
Lainé, Atomic proximity between S4 segment and pore domain in Shaker potassium channels. 2003, Pubmed , Xenbase
Lee, Two separate interfaces between the voltage sensor and pore are required for the function of voltage-dependent K(+) channels. 2009, Pubmed , Xenbase
Lee, Functional reconstitution of purified human Hv1 H+ channels. 2009, Pubmed
Li, Structural basis of lipid-driven conformational transitions in the KvAP voltage-sensing domain. 2014, Pubmed
Li, Resting state of the human proton channel dimer in a lipid bilayer. 2015, Pubmed
Li, Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain. 2014, Pubmed , Xenbase
Li, Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. 1992, Pubmed
Li-Smerin, A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel. 2000, Pubmed , Xenbase
Long, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. 2005, Pubmed
Long, Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. 2007, Pubmed
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Lu, Coupling between voltage sensors and activation gate in voltage-gated K+ channels. 2002, Pubmed , Xenbase
Lu, Ion conduction pore is conserved among potassium channels. 2001, Pubmed
Mannuzzu, Direct physical measure of conformational rearrangement underlying potassium channel gating. 1996, Pubmed , Xenbase
Moreau, Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy. 2015, 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
Musset, Aspartate 112 is the selectivity filter of the human voltage-gated proton channel. 2011, Pubmed
Männikkö, Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties. 2005, Pubmed , Xenbase
Olcese, Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels. 1997, Pubmed , Xenbase
Perozo, Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. 1993, Pubmed
Petitjean, A Disease Mutation Causing Episodic Ataxia Type I in the S1 Links Directly to the Voltage Sensor and the Selectivity Filter in Kv Channels. 2015, Pubmed , Xenbase
Piper, Gating currents associated with intramembrane charge displacement in HERG potassium channels. 2003, Pubmed , Xenbase
Priest, S3-S4 linker length modulates the relaxed state of a voltage-gated potassium channel. 2013, Pubmed
Ramsey, A voltage-gated proton-selective channel lacking the pore domain. 2006, Pubmed
Sali, Comparative protein modelling by satisfaction of spatial restraints. 1993, Pubmed
Sasaki, A voltage sensor-domain protein is a voltage-gated proton channel. 2006, Pubmed
Shen, Deletion analysis of K+ channel assembly. 1993, Pubmed , Xenbase
Starace, A proton pore in a potassium channel voltage sensor reveals a focused electric field. 2004, Pubmed
Taglialatela, Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes. 1992, Pubmed , Xenbase
Takeshita, X-ray crystal structure of voltage-gated proton channel. 2014, Pubmed
Tan, Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels. 2012, Pubmed , Xenbase
Tao, A gating charge transfer center in voltage sensors. 2010, Pubmed , Xenbase
Tombola, Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores. 2005, Pubmed , Xenbase
Tombola, The twisted ion-permeation pathway of a resting voltage-sensing domain. 2007, Pubmed , Xenbase
Vargas, An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations. 2012, Pubmed
Villalba-Galea, S4-based voltage sensors have three major conformations. 2008, Pubmed
Wall-Lacelle, Double mutant cycle analysis identified a critical leucine residue in the IIS4S5 linker for the activation of the Ca(V)2.3 calcium channel. 2011, Pubmed
Yarov-Yarovoy, Structural basis for gating charge movement in the voltage sensor of a sodium channel. 2012, Pubmed
del Camino, Status of the intracellular gate in the activated-not-open state of shaker K+ channels. 2005, Pubmed , Xenbase