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	Fig. 1. Effect of split position displacement on activation voltage dependence of Kv11.1 split channels. a Kv11.1 sequence of the S4–S5 linker region. Linker boundaries are chosen based in the structural information provided by the cryo-EM architecture of Kv11.1 [60]. b Representative membrane currents from individual oocytes expressing the indicated channel variants submitted to 1-s depolarization pulses at different potentials at 10 mV intervals form a holding potential of − 80/− 100 mV, followed by a repolarization step as indicated at the top. Note the different repolarization potentials for different constructs as enclosed in parentheses at the end of the current traces. Currents recorded without leak subtraction are shown. WT continuous wild type. c Averaged I versus V relationships measured at the end of the depolarization step (signalled by the circle in the WT traces of b). Note the typical Kv11.1 n-shaped curves due to the strong rectification as a result of the slow activation and fast inactivation overlap at positive voltages. d Plots of normalized peak tail current magnitudes (square in the WT current traces of b) as a function of depolarizing voltage. Continuous lines are Boltzmann fits to the data as indicated in “Materials and methods” section
	Fig. 2. Effect of split position displacement on Kv11.1 split voltage-dependent activation rates. a Comparison of time course of current activation at 0 mV. Families of representative membrane currents from individual oocytes (left). The duration of a depolarizing prepulse to 0 mV was varied as illustrated in the envelope-of-tail currents protocol at the top, illustrative of that used with WT channels, followed by a repolarization step to the indicated potentials (− 70, − 50, − 80, − 120 and − 140 mV for WT, split 545, split 541, split 540 and split 539 variants, respectively). Cells expressing split 539 and split 540 channels were held at − 140 and − 120 mV trying to ensure that they also start in deactivated state. An enhanced view of the very rapidly deactivating split 545 tail current at the end of the 2560-ms depolarizing step is shown in the inset. Averaged plots of normalized tail current magnitudes versus depolarization time at 0 mV (right). Values from continuous wild-type channels are shown as a dotted line for comparison. An expansion of the initial 200 ms to highlight the progressive disappearance of the early current delay in the sigmoidal activation time course is shown in the inset. b Comparison of activation rate voltage dependence. Plots of normalized tail current magnitudes versus depolarization times at voltages between − 30 and + 40 mV (split 545) or − 60 and + 40 mV (split 540) at 10 and 20 mV intervals were generated from current traces as indicated in a (left). These plots were used to measure times necessary to attain half-maximum current magnitudes (dotted lines). Expanded views of the initial 250 ms of the plots are shown in the insets. Data obtained at 0 mV are represented in black. Dependence of activation rates on depolarization membrane potentials (right). The time necessary to attain half-maximum tail current magnitude is plotted versus depolarization potential. Values from continuous wild-type channels are shown as a dotted line for comparison
	Fig. 3. Effect of split position displacement on voltage-dependent deactivation kinetics of Kv11.1 splits. 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” section. Only the magnitude of the deactivation time constant corresponding to the fast decaying major component of current at negative voltages is shown
	Fig. 4. Functional properties of split channels carrying partial S4-S5 linker deletions around the disconnection point between the VSD and the PD. a Activation and deactivation gating kinetics of split 539 channels lacking Asp540 or Asp540 plus Arg541 residues. Activation voltage dependence (top). Fractional activation curves were obtained from oocytes co-expressing 1–539 plus 541–1159 or 1–539 plus 542–1159 demi-channel combinations (that will lack D540 or D540 plus R541 residues, respectively), using tail current data as detailed in Fig. 1 for split 539. Continuous lines are Boltzmann curves that best fitted the data as indicated in “Materials and methods” section. A Boltzmann curve from non-deleted split 539 channels as in Fig. 1d is also shown for comparison. Analogy of deactivation kinetics between L539 split channels with and without residues D540 and D540 plus R541 (bottom left). Deactivation time constants were quantified from double exponential fits to the tails as in Fig. 3 (see also “Materials and methods” section). Plots of deactivation time constants for the fast decaying component of the currents as a function of membrane potential are shown. Data from 1 to 539 plus 541–1159 (squares) and 1–539 plus 542–1159 (circles) demi-channel combinations appear superimposed to those from non-deleted split 539 channels (dotted line). Data from continuous wild-type and split 545 channels are also shown as dashed lines for comparison. Comparison of activation rates at 0 mV (bottom right). Averaged plots of normalized tail current magnitudes versus depolarization time at 0 mV are shown from 1 to 539 plus 541–1159 (squares) and 1–539 plus 542–1159 (circles) demi-channel combinations. An expansion of the initial 200-ms time course is shown in the inset. Data from continuous wild-type (dashed line) and non-deleted split 539 channels (dotted line) are also shown for comparison. b Activation and deactivation gating kinetics of split 545 channels lacking either Tyr545 or the Ser543 + Glu544 + Tyr545 segment. Analysis of activation voltage dependence (top), deactivation kinetics (bottom left) and the time course of current activation at 0 mV (bottom right) were performed as detailed in a. Data from oocytes expressing the 1–544 plus 546–1159 (lacking only the Y545 residue, circles) and the 1–542 plus 546–1159 (lacking the S543 + E544 + Y545 triplet, squares) demi-channel combinations are depicted. Due to the low level of expression obtained with the Y545-deleted construct, a high-K+ extracellular solution was used for the 1–542 plus 546–1159 combination recordings (see “Materials and methods” section). Data from continuous wild-type (dotted lines) and non-deleted split 545 channels (dashed lines) are also shown. In both cases, the traces indicating the slowest time constants correspond to data obtained in high-K+ extracellular solution
	Fig. 5. Deactivation kinetics of channels split at the beginning or the end of the S4–S5 linker are not modified by a mutation in residue 3 of the Kv11.1 N-tail. a Deactivation characteristics of split 545 channels carrying a V3C mutation in the N-tail of the N-terminal demi-channel. A representative family of currents is shown on the left, obtained with the protocol shown on top of the currents. A high-K+ extracellular solution was used to maximize the currents due to the small current magnitudes obtained with the split 545 + V3C construct (see “Materials and methods” section). Only the first part of the 4-s repolarization steps is shown. An expanded view of the initial part of the tails is shown in the inset. The dependence of deactivation rates on repolarization membrane potential is shown on the right. Only the magnitude of the deactivation time constant corresponding to the fast decaying major component of current at negative voltages is shown. Data from WT, the full-length V3C mutant and non-mutated split 545 channels also recorded in high-K+ are shown for comparison as indicated. b Deactivation characteristics of split 540 channels carrying a V3C mutation in the N-tail of the N-terminal demi-channel. A representative family of currents is shown on the left. The dependence of deactivation rates on repolarization membrane potential is shown on the right. Data from WT, full-length V3C and non-mutated split 540 and split 545 channels are also shown for comparison
	Fig. 6. Effect of S4–S5 linker interruptions on Kv11.1 ionic current mode shift. a Representative ion currents from continuous wild-type (WT) and splits 545 and 539 studied with the voltage protocols shown at − 80/− 100 (top) and + 40 mV (bottom) holding potentials. b Averaged I versus V relationships from currents measured at the end of the variable depolarization step at different voltages as in panel a. Open and closed symbols correspond to data obtained in the same oocytes at hyperpolarized and depolarized holding potentials, respectively. c Plots of normalized peak tail current magnitudes as a function of depolarizing voltage for channels split at different positions along the S4–S5 linker. Data from continuous WT channels are also shown for comparison. Averaged values from four to eight cells are shown. Sometimes, error bars are smaller than the symbols. Continuous lines are Boltzmann fits to the data as indicated in “Materials and methods” section. Hyperpolarized (open symbols) and depolarized (closed symbols) holding potential values are indicated in the graphs
	Fig. 7. Effect of split position displacement on voltage dependence of MTSET availability to an engineered cysteine at position 521 in the extracellular part of the voltage sensor S4 helix. Summary of voltage dependence of the MTSET effect in different splits carrying the I521C mutation at the extracellular part of the voltage sensor S4 helix, as a function of the holding potential used during the MTS reagent exposure (upper panels). Plots of voltage dependence of the MTSET effect in the individual splits tested (lower panels). Magnitudes of MTSET-induced variations in current kinetics during test ramps as indicated in “Materials and methods” section following a 2-min exposure to 1 mM of the 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 correspond to fits using a Boltzmann function as indicated in “Materials and methods” section. The corresponding V 1/2 values are shown on the graphs. G/V plots from the same constructs obtained from fits to tail current data and V 1/2 values derived from them are also shown in red. Note the similarity of these plots and those from the same splits without the I521C plus C445V and C449V mutations (gray dotted lines; reproduced from Fig. 1d)
	Fig. 8. Close apposition and interactions of the Kv11.1 N-tail with the S4–S5, S2–S3 and C-linkers of the channel. a Lateral view of the Kv11.1 tetrameric structure ([60] PDB code 5VA2). A highlighted subunit is coloured in gray. b Enhanced view of the region inside the signalled square in a with ribbons corresponding to the indicated domains and residue symbols coloured as shown at the bottom. Lateral chains of the selected residues are shown as ball and stick with oxygen and nitrogen atoms as red and blue spheres. Dashed lines indicate interatomic distances ranging between 3.0 and 5.4 Å. Structures were processed with UCSF Chimera [39]

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