de la Peña P et al. (2018), Functional characterization of Kv11.1 (hERG) po...

XB-ART-56254

Pflugers Arch 2018 Jul 01;4707:1069-1085. doi: 10.1007/s00424-018-2135-y.

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Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

de la Peña P , Domínguez P , Barros F .

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Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

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Species referenced: Xenopus laevis
Genes referenced: dtl kcnh2
GO keywords: potassium channel activity

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	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

References [+] :

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