de la Peña P , Domínguez P , Barros F .

	Fig. 1 Tridimensional structure of a Kv11.1 subunit transmembrane core region and position of the split points at the different extra and intracellular loops linking the transmembrane segments. A lateral view of the Kv11.1 tetrameric structure ([65]; PDB code 5VA2) with the subunit corresponding to the enhanced view in the main panel highlighted in gray, is shown on the lower right. The region coloured in gray in the central panel corresponds to the pore domain including the S5 and S6 helices. The S4–S45 linker is in magenta. Ribbons from the S4, S3, S2 and S1 segments are in yellow, cyan, brown and green, respectively. The position of the residues providing the positive charges to the S4 helix is marked in orange. Lateral chains of residues 478 and 482 at which the S2–S3 linker was sectioned in this study, and 510 and 521 that mark the boundaries of the structurally undefined S3–S4 linker are shown as ball and stick. The location of the different split points described in this study are indicated, with those that did not yield functional expression marked with open arrows and those giving rise to functional channels marked with solid arrows. A view of the extracellular and structurally defined S1–S2 and S3–S4 linkers region from the homologous Kv10.1 channel ([67]; PDB code 5K7L) is depicted in the upper inset, showing the corresponding position of the Kv11.1514 and 518 splits. Structures were processed with UCSF Chimera [41]
	Fig. 2 Characterization of activation gating voltage dependence of S2–S3 linker splits. a Split 478. Representative membrane currents from individual oocytes submitted to 1 s depolarization pulses to different potentials at 10 mV intervals from a negative (− 80 mV, left) and a positive (+ 40 mV, right), followed by a repolarization step to − 50 mV. Currents recorded without leak subtraction are shown. Averaged I vs V relationships measured at the end of the depolarization step (signalled by the solid square at the top) and at the peak of the tail current (G/V plots, circles) are shown at the bottom. Continuous lines in the G/V plots are Boltzmann fits to the data as indicated in ‘Materials and methods’. A G/V plot from continuous wild-type channels (WT, dashed line) is also shown for comparison. A family of wild-type channel currents in response to 1-s depolarizing pulses from − 80 to + 40 mV, followed by a repolarization to − 70 mV, is shown in the inset. In this case, the horizontal and vertical scale bars correspond to 200 ms and 500 nA, respectively. b Split 482. Recording of currents in response to the protocols shown on top of the traces and analysis were performed as detailed in A
	Fig. 3 Characterization of voltage-dependent activation rates of S2–S3 linker splits. a Time course of current activation at 0 mV. Families of representative membrane currents from individual oocytes are shown. The duration of a depolarizing prepulse to 0 mV was varied as illustrated in the envelope-of-tail-currents protocol at the top, followed by a repolarization step to the indicated potentials. Cells expressing wild-type and split 478 channels were held at − 80 mV and those expressing split 482 channels at − 100 mV to ensure that they always started in a deactivated state. b Averaged plots of normalized tail current magnitudes vs depolarization time at different potentials. Most error bars are smaller than the symbols. Values from continuous wild-type channels at 0 mV are shown as a dotted line for comparison. Expansions of the initial tens of ms to highlight the early current delay in the sigmoidal activation time courses are shown in the insets. c Comparison of activation rate voltage-dependence. Plots of normalized tail current magnitudes vs depolarization times (a) were used to measure times necessary to attain half-maximum current magnitudes, that are plotted vs depolarization potential. Values from continuous wild-type channels (WT) are shown as a dotted line for comparison
	Fig. 4 Effect of S2–S3 linker splits on Kv11.1 voltage-dependent deactivation kinetics. Representative families of currents are shown at the top, obtained during steps to potentials ranging from − 20 to − 140 mV in 10-mV intervals, following depolarization pulses at + 40 mV to open (and inactivate) the channels, using the indicated protocol. For clarity, only the first part of the 4-s repolarization steps used to follow the complete decay of the tail currents is shown. The dependence of deactivation rates on repolarization membrane potential is shown at the bottom. Deactivation time constants were quantified by fitting a double exponential to the decaying portion of the tails as described in ‘Materials and methods’. Fast (circles) and slow (squares) deactivation time constants are depicted in the plot. Data corresponding to the fast decaying component from continuous wild-type channels (WT) are shown as a dotted line for comparison
	Fig. 5 Effect of S2–S3 linker interruptions on voltage dependence of MTSET availability to an engineered cysteine at position 521 in the extracellular part of the voltage sensor S4 helix. a, b Absence of MTSET effects in cells held at negative potentials not submitted to repetitive depolarization pulsing, and determination of the voltage range at which the MTS reagent induces its effects under steady-state conditions. Current traces obtained in response to the indicated voltage protocols are shown for 478 (a) and 482 (b) splits. No pulses were applied during the 2-min periods indicated by black boxes at which the cells were continuously held at the indicated potential. A high K+ extracellular solution was used to maximize the currents due to the small current magnitudes obtained with the Split 482 construct (see ‘Materials and methods’). Arrowheads are used to indicate the time at which the current variations were estimated quantifying either the magnitude of the final versus peak current during the voltage steps following the ramp pulses or the rectification factor during the ramps as indicated in Methods. Similar results were obtained in both cases. c, d Voltage dependence of the MTSET effect. Magnitudes of MTSET-induced variations in currents measured (b), following a 2-min exposure to 1 mM of MTS reagent without pulsing at the holding potentials indicated in the abscissa were normalized to those observed at a positive potential value of + 40 mV. Due to the irreversibility of the MTSET effects, only one reagent application was performed and a single holding potential value (followed by a positive control at + 40 mV) was checked in each cell studied. Data from three to six cells were averaged for every single point. Some error bars are smaller than the symbols. Continuous lines linking the data points correspond to fits using a Boltzmann function as indicated in ‘Materials and methods’. The corresponding V1/2 values are shown on the graphs. G/V plots from the same constructs obtained from fits to tail current data and V1/2 values derived from them are also shown as indicated. Note the similarity of these plots and those from the same splits without the mutations I521C, C445V and C449V (dotted lines; reproduced from split channels curves in Fig. 2) introduced to study the MTSET-induced effects (see ‘Materials and methods’ section)
	Fig. 6 Effect of S2–S3 linker interruptions on MTSET-induced modification rates of engineered cysteine 521. a Split 478. Current traces shown at the top were obtained in response to the indicated protocol that was repeated at 5-s intervals. Current traces corresponding to times immediately before application of 1 mM MTSET and at the end of the MTSET exposure using hyperpolarizing (− 100 mV) and depolarizing (+ 40 mV) holding potentials are highlighted as thicker black lines. The bottom plot illustrates the time course of MTSET-induced modifications normalized to those observed at the end of the treatment. The changes in the peak/end current relationship during the − 120 mV voltage step were used to generate the plot. Mono-exponential fits to the data are shown superimposed on the symbols. The values of the corresponding time constants (tau) are indicated in the graphs. b Split 482. Plots represent the representative currents and time course of MTSET-induced modifications as detailed (a). A high K+ extracellular solution was used with the Split 482 construct
	Fig. 7 Activation and deactivation gating characteristics of Kv11.1 channels split at the S3–S4 linker. a Voltage dependence of activation gating. Representative membrane currents from an oocyte expressing the 514 split channel are shown on the left. Currents were recorded using the protocols shown on top of the traces at − 80 and + 40 mV holding potentials as indicated. Currents are shown without leak subtraction. Averaged I vs V relationships measured at the end of the depolarization step (signalled by the solid circle on the left) and at the peak of the tail current (G/V plots, squares on the left) are shown in the right panels. In this case, not only data from split 514 (circles and squares), but also those obtained from split 518 (triangles) are depicted. Data from continuous wild-type channels obtained at a holding potential of − 80 mV are also shown as dotted lines for comparison. b Comparison of activation rates at 0 mV. Representative membrane currents from an oocyte expressing the 514 split channel are shown on the left. Averaged plots of normalized tail current magnitudes versus depolarization time at 0 mV are shown on the right both for 514 (circles) and 518 (triangles) splits. Values from continuous wild-type channels are shown as a dotted line for comparison. An expansion of the initial tens of ms to highlight the early current delay in the sigmoidal activation time course is shown in the inset. c Voltage-dependent deactivation kinetics. A representative family of currents from an oocyte expressing the 514 split channel is shown on the left. The dependence of deactivation rates on repolarization membrane potential is shown on the right. Fast (closed symbols) and slow (open symbols) deactivation time constants for split 514 (circles) and split 518 (triangles) splits are depicted in the plot. Data corresponding to the fast decaying component from continuous wild-type channels are also shown as a dotted line for comparison
	Fig. 8 Effect of S3–S4 linker interruptions on MTSET availability to an engineered cysteine at position 521 in the extracellular part of the voltage sensor S4 helix. a Steady-state voltage dependence of MTSET effects. Representative current traces from two oocytes expressing 514 split channels held at the indicated potentials and not submitted to repetitive depolarization pulsing are illustrated at the top. No pulses were applied during the 2-min periods indicated by black boxes at which the cells were continuously held at the indicated potential. Measurements of current variations at the end of the repolarizing step (circles) were used to determine the voltage range at which the MTS reagent induces its effects under steady-state conditions as shown at the bottom. Plots obtained from split 514 (bottom left) and split 518 channels (bottom right) are shown. Continuous lines linking the data points correspond to fits using a Boltzmann function as indicated in ‘Materials and methods’. The corresponding V1/2 values are shown on the graphs. G/V plots from the same constructs obtained from fits to tail current data and V1/2 values derived from them are also shown. Note the similarity of these plots and those from the same splits without the mutations I521C, C445V and C449V (dotted lines; reproduced from Fig. 7a). bMTSET-induced modification rates of cysteine 521. Current traces shown at the top were obtained in response to the indicated protocols that were repeated at 5-s intervals. Data from 514 (left) and split 518 channels (right) held at − 100 and + 40 mV are shown as indicated. A high K+ extracellular solution was used to maximize the currents of the Split 514 construct. Current traces corresponding to times immediately before application of 1 mM MTSET and at the end of the MTSET exposure are highlighted as thicker black lines. Plots of the time course of MTSET-induced modifications normalized to those observed at the end of the treatment are depicted at the bottom. The changes in the peak/end current relationship during the − 120 mV voltage steps were used to generate the plots. The time of MTSET introduction into the recording chamber is marked with an arrow. Mono-exponential fits to the data are shown superimposed to the symbols. Data from I521C full-length continuous channels at − 100 and + 40 mV are shown for a better comparison as dashed and dotted lines, respectively. Note the different time scale of the split 514 and split 518 plots in the abscissa

Alonso-Ron, Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions. 2008, Pubmed, Xenbase

Alonso-Ron, Thermodynamic and kinetic properties of amino-terminal and S4-S5 loop HERG channel mutants under steady-state conditions. 2008, Pubmed , Xenbase
Anderson, Phylogeny of ion channels: clues to structure and function. 2001, Pubmed
Arrigoni, The voltage-sensing domain of a phosphatase gates the pore of a potassium channel. 2013, Pubmed , Xenbase
Babcock, hERG channel function: beyond long QT. 2013, Pubmed
Barros, Cytoplasmic domains and voltage-dependent potassium channel gating. 2012, Pubmed
Barros, Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C. 1998, Pubmed , Xenbase
Bezanilla, How membrane proteins sense voltage. 2008, Pubmed
Bichet, Merging functional studies with structures of inward-rectifier K(+) channels. 2003, Pubmed
Blunck, Mechanism of electromechanical coupling in voltage-gated potassium channels. 2012, Pubmed
Castillo, Voltage-gated proton (H(v)1) channels, a singular voltage sensing domain. 2015, Pubmed
Cheng, Voltage-dependent gating of HERG potassium channels. 2012, Pubmed
Decoursey, Voltage-gated proton channels. 2012, Pubmed
de la Peña, Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains. 2018, Pubmed , Xenbase
de la Peña, Mapping of interactions between the N- and C-termini and the channel core in HERG K+ channels. 2013, Pubmed , Xenbase
de la Peña, Interactions between the N-terminal tail and the gating machinery of hERG K⁺ channels both in closed and open/inactive states. 2015, Pubmed , Xenbase
de la Peña, Demonstration of physical proximity between the N terminus and the S4-S5 linker of the human ether-a-go-go-related gene (hERG) potassium channel. 2011, Pubmed , Xenbase
Dobrzynski, Structure, function and clinical relevance of the cardiac conduction system, including the atrioventricular ring and outflow tract tissues. 2013, Pubmed
Dou, The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium. 2013, Pubmed , Xenbase
Es-Salah-Lamoureux, Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening. 2010, Pubmed , Xenbase
Feliciangeli, The family of K2P channels: salient structural and functional properties. 2015, Pubmed
Fernández-Trillo, Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating. 2011, Pubmed
Gómez-Varela, Influence of amino-terminal structures on kinetic transitions between several closed and open states in human erg K+ channels. 2002, Pubmed , Xenbase
González, K₂p channels in plants and animals. 2015, Pubmed
Goodchild, Sequence of gating charge movement and pore gating in HERG activation and deactivation pathways. 2015, Pubmed , Xenbase
Goodchild, Gating charge movement precedes ionic current activation in hERG channels. 2014, Pubmed
Gustina, A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels. 2009, Pubmed , Xenbase
Hibino, Inwardly rectifying potassium channels: their structure, function, and physiological roles. 2010, Pubmed
Isacoff, Conduits of life's spark: a perspective on ion channel research since the birth of neuron. 2013, Pubmed
Jegla, Evolution of the human ion channel set. 2009, Pubmed
Kiehn, Pathways of HERG inactivation. 1999, Pubmed , Xenbase
Liu, Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes. 1996, Pubmed
Lörinczi, Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains. 2015, Pubmed , Xenbase
Lu, Ion conduction pore is conserved among potassium channels. 2001, Pubmed
Matsuura, Rapidly and slowly activating components of delayed rectifier K(+) current in guinea-pig sino-atrial node pacemaker cells. 2002, Pubmed
Mishima, The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel. 2016, Pubmed
Ng, Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation. 2016, Pubmed , Xenbase
Ng, The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation. 2011, Pubmed
Ng, The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating. 2012, Pubmed
Niemeyer, Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia? 2016, Pubmed
Pardo, The roles of K(+) channels in cancer. 2014, Pubmed
Pettersen, UCSF Chimera--a visualization system for exploratory research and analysis. 2004, Pubmed
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
Sand, Fine-tuning of voltage sensitivity of the Kv1.2 potassium channel by interhelix loop dynamics. 2013, Pubmed , Xenbase
Sanguinetti, Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes. 1999, Pubmed , Xenbase
Sanguinetti, HERG1 channelopathies. 2010, Pubmed
Sanguinetti, hERG potassium channels and cardiac arrhythmia. 2006, Pubmed
Sato, Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1. 2003, Pubmed , Xenbase
Sato, Integration of Shaker-type K+ channel, KAT1, into the endoplasmic reticulum membrane: synergistic insertion of voltage-sensing segments, S3-S4, and independent insertion of pore-forming segments, S5-P-S6. 2002, Pubmed , Xenbase
Schmidt-Rose, Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1. 1997, Pubmed , Xenbase
Subbiah, Molecular basis of slow activation of the human ether-a-go-go related gene potassium channel. 2004, Pubmed , Xenbase
Swartz, Sensing voltage across lipid membranes. 2008, Pubmed
Syeda, Tetrameric assembly of KvLm K+ channels with defined numbers of voltage sensors. 2012, Pubmed
Tombola, Architecture and gating of Hv1 proton channels. 2009, Pubmed
Tomczak, A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore. 2017, Pubmed , Xenbase
Trudeau, HERG, a human inward rectifier in the voltage-gated potassium channel family. 1995, Pubmed
Tu, Transmembrane biogenesis of Kv1.3. 2000, Pubmed , Xenbase
Vandenberg, hERG K(+) channels: structure, function, and clinical significance. 2012, Pubmed
Vardanyan, Coupling of voltage-sensors to the channel pore: a comparative view. 2012, Pubmed
Villalba-Galea, Voltage-Controlled Enzymes: The New JanusBifrons. 2012, Pubmed
Viloria, Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating. 2000, Pubmed , Xenbase
Wang, Regulation of deactivation by an amino terminal domain in human ether-à-go-go-related gene potassium channels. 1998, Pubmed , Xenbase
Wang, Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels. 2000, Pubmed , Xenbase
Wang, Cryo-EM Structure of the Open Human Ether-à-go-go-Related K+ Channel hERG. 2017, Pubmed
Wang, Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels. 2013, Pubmed
Wang, A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes. 1997, Pubmed , Xenbase
Whicher, Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism. 2016, Pubmed
Yellen, The voltage-gated potassium channels and their relatives. 2002, Pubmed
Yu, The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. 2004, Pubmed
Zhang, Contribution of hydrophobic and electrostatic interactions to the membrane integration of the Shaker K+ channel voltage sensor domain. 2007, Pubmed