Lörinczi É , Gómez-Posada JC , de la Peña P , Tomczak AP , Fernández-Trillo J , Leipscher U , Stühmer W , Barros F , Pardo LA .

	Figure 1. Discontinuous KV10.1 channels give rise to voltage-dependent currents.(a) Schematic representation of KV10.1 deletions. The voltage-sensing domain is depicted in green, the S4–S5 linker in red and the pore module in dark blue. (b) Families of current traces obtained by depolarizations to different potential (up to +80 mV at 20 mV intervals) from a holding voltage of −80 mV in oocytes injected with cRNA for continuous KV10.1 (left), a mixture of cRNAs encoding for residues 1–347 and 348–962 (centre) or residues 1–341 and 350–962 (right). All gave rise to outward currents with similar kinetics, but with some differences in their voltage dependence as illustrated in e. Extracellular solution containing 2.5 mM KCl and no MgCl2 was used. (c). Representative families of currents for continuous and 347-split KV10.1 channels recorded in 60 mM extracellular K+. The tail amplitude at time 0 (represented in e) was extrapolated from biexponential fits as described in Methods and as indicated by symbols. (d) Families of currents elicited in 60 mM extracellular K+ using oocytes injected with cRNA encoding 1–347 (left) or 348–962 (right) KV10.1 demi-channels. Note the lack of currents after injection of each of the demi-channels alone as compared with the continuous and the split channels (lower panels; scale bars, 2 μA, 100 ms). (e). Conductance/voltage plot of continuous (open circles, n=13), 347-split (closed circles, n=12) and split KV10.1 channels lacking the S4–S5 linker (open squares, n=11). The currents elicited by channels completely lacking the S4–S5 segment required stronger depolarizations to develop, but the slope (that is, voltage dependence) was not dramatically different. Error bars represent s.e.
	Figure 2. Both KV10.1 demi-channels are detected in the same complex.(a) Biotinylation of surface proteins and subsequent pull down of labelled molecules allowed the detection of full-length channel using an anti-KV10.1 C-terminal antibody, and also of the C-terminal truncated protein when expressed either alone or as split channel. (b) Co-immunoprecipitation of N- and C- terminal demi-channels. The N-terminal fragment was labelled with 5 × Myc and the C-terminal was 4 × HA tagged. Immunoprecipitation with HA-tag pulled down a fragment of size compatible with the N-terminal demi-channel (arrow), recognized by anti-Myc immunoblot. Asterisks indicate bands corresponding to the antibody used to immunoprecipitate. (c) Immunoprecipitation with anti-Myc also pulled HA-tagged fragments detected as a double band (arrows). The migration distance of the upper band was modified by deglycosylation (PNGase F lanes), as expected for the C-terminal fragment of KV10.1, which contains the glycosylated residues. Input lanes were loaded with the extract corresponding to half an oocyte; the equivalent to 30 oocytes were used to immunoprecipitate. (d) Native electrophoresis and immunoblot shows the presence of a complex with size similar to that of the continuous channel when demi-channels were expressed together, recognized by both anti-Myc and anti-KV10.1 antibodies.
	Figure 3. Split channels retain dependence on the prepulse potential and on extracellular Mg2+.(a,b) Representative current traces obtained following the depicted protocol, holding the oocyte for 5 s (indicated in the schematic as a dotted line) at the indicated potential and immediately stimulating to +40 mV in the absence (upper traces) or the presence (middle traces) of 5 mM extracellular Mg2+. Both wild-type (a) and split (b) channels show accelerated activation at less hyperpolarized potentials. Also in both cases, Mg2+ slows down the activation. The effect of prepulse potential at different Mg2+ concentrations is depicted in the lowest panels (n=5–8). Over 1 mM, the activation of the split channel is so slow that the dependence on prepulse is not any longer evident. The solid lines indicate fits to sigmoid functions, dotted lines represent polynomial fits. (c) Comparison of data (from a,b) of continuous and split channel in 1 mM Mg2+. (d) Effect of Mg2+ concentration on the speed of activation of continuous and split channels. The activation was slower in the split channels (closed symbols) than in control (open symbols) under all conditions tested. Error bars represent s.e.
	Figure 4. Split channels retain properties residing in both the voltage-sensing and permeation modules.(a) Mutation of the voltage-sensor domain has similar effects in continuous and split channels. Neutralization of a positive charge in the S4 domain (R336Q) shifts the activation potential to hyperpolarized values regardless of the integrity of the S4–S5 linker (Open circles, wild type; closed circles, split channel; open diamonds, R336Q; closed diamonds, split R336Q; n=7; error bars, s.e.). (b) Blockade by astemizole, whose structural determinants lie in the C-terminal part of the protein, is conserved both in channels with interrupted S4–S5 (closed circles, n=6–7) or lacking completely the linker (squares, n=2–4), albeit with reduced affinity as compared with control (open circles, n=9–16). (c–e) A pore mutation precluding K+ permeation abolishes the activity also of split channels (c, wild-type split; d, G440S mutant split) (e) I/V relationship of wild-type and mutant split channels constructed with values obtained from 10 oocytes. Error bars represent s.e.
	Figure 5. Interruption of the S4–S5 linker renders also functional KV10.2 channels.Current amplitudes were always bigger in continuous (a) than split (b) channels. The split channel (solid symbols, n=4 for this plot) also required larger depolarization than the continuous channel (open symbols, n=14) to be activated (c). Error bars represent s.e.
	Figure 6. Functional expression and activation kinetics of continuous and Y545 Split KV11.1 (HERG) channels.(a) Representative current traces of continuous wild-type HERG channels in response to the protocol shown at the top. Extracellular medium containing 2 and 50 mM KCl was used as indicated. (b) Voltage-dependent activation of Y545 Split KV11.1 channels. Left, raw current traces recorded in 2 and 50 mM extracellular K+ in response to 1 s depolarization pulses between −70 and +60 mV at 10 mV intervals from a holding potential of −80 mV, followed by a repolarizing step to −40 (2 mM K+) and −70 (50 mM K+) mV. Centre, averaged I versus V relationship measured at the end of the depolarization step in 2 mM external K+ (n=8). Note the typical n-shaped curve due to the strong rectification as a result of the typical KV11.1 slow activation and fast inactivation overlap at positive voltages. Right, plots of normalized peak tail currents as a function of depolarizing voltage in 2 (closed circles) and 50 mM (open circles) extracellular K+ (n=13). A curve from wild-type continuous KV11.1 channels obtained in the same conditions is shown as a dashed line for comparison. (c) Y545 Split KV11.1 voltage-dependent activation rates. Left, time course of current activation at 0 mV in 2 (top traces) and 50 mM (lower traces) extracellular K+. The duration of a depolarizing prepulse from a holding potential of −80 mV was varied and followed by a repolarization step to −50 and −100 mV in 2 and 50 mM external K+, respectively. An enhanced view of the tail currents at the end of the 2,560 ms depolarizing steps is shown in the insets. Centre, plot of normalized tail current magnitude versus depolarization time at 0 mV (n=4). The plot was used to measure time necessary to attain half-maximum current magnitude (dashed arrow). Note the sigmoidal nature of the early activation time course during the initial tens of ms. Right, dependence of activation rates on depolarisation membrane potential (n≥4). Values from wild-type non-split channels are shown as a dashed line for comparison. Error bars represent s.e.
	Figure 7. Characterization of deactivation and inactivation properties of Y545 Split KV11.1 channels.(a) Voltage-dependent deactivation. Top panels, representative current traces recorded in 50 mM extracellular K+ during steps to potentials ranging from −20 to −120 mV at 10 mV intervals, following depolarizing pulses to +40 mV from a holding potential of −80 mV as schematized at the top. Currents from oocytes expressing continuous wild-type (left) and Y545 Split KV11.1 (right) are shown for comparison. For the split, also an enhanced view of the peak tail currents during the repolarization steps is shown in the inset. Lower panel, plot of fast deactivation time constant for different repolarization voltages for split (open circles, n=4) and wild-type non-split channels (dashed line). Error bars, s.e. (b) Measurement of Y545 Split KV11.1 inactivation rates. Left panel, onset of fast inactivation at different voltages was studied in 2 mM extracellular K+ with the triple pulse protocol shown at the top, in which the channels were activated and inactivated with a 1 s prepulse to +40 mV, followed by a second short prepulse to −100 mV to recover the channels from inactivation and a test pulse to different voltages from −60 to +100 mV at 10 mV intervals to reinactivate them. Membrane currents starting at the end of the depolarizing prepulse for depolarizations between +20 and +100 mV at which the inactivation and deactivation kinetics barely overlap, are shown in the inset. Right panel, plot of time constants for the onset of inactivation in 2 mM (closed circles) and 50 mM (open circles) extracellular K+ as a function of voltage, obtained from single-exponential fits to the decaying portion of the currents during the test pulses. Values from wild-type non-split channels at the same K+ concentrations are shown as black (2 mM) and grey (50 mM) dashed lines for comparison.
	Figure 8. Y545 Split HERG channels retain properties residing in the permeation module.(a) Inhibition of split HERG currents by E-4031. The time course of current inhibition in response to an addition of 100 μM E-4031 is shown on the left. Currents were elicited in response to 1 s depolarizing pulses to +40 mV from a holding potential of −40 mV followed by a repolarization step to −50 mV. Pulses were delivered at a frequency of 0.1 Hz. Representative tail current traces before and after 5 min of treatment with E−40131 are shown on the insets. A bar histogram relating the extent of current inhibition with the concentration of the inhibitor is shown on the right. (b). Absence of functional expression upon co-injection of cRNAs encoding N-terminal 1–545 and non-conducting C-terminal 546–1,159 KV11.1 demi-channels carrying a G628S point mutation in the pore. Currents were obtained in 50 mM extracellular K+ using the voltage protocol shown at the top. Families of currents from non-mutant (left) and G628S (right) splits are shown. (c) Characterization of voltage-dependent currents elicited by co-expression of KV11.1 demi-channels carrying a S620T point mutation in the pore domain. The voltage protocol used to elicit the currents is shown at the top. Representative families of currents recorded in response to the protocol are shown in the insets. Top panels, I versus V relationships from normalized currents measured in 2 mM (open squares, left) and 50 mM (closed circles, right) extracellular K+ at the end of the depolarization step. Lower panel, current/voltage relationship generated using peak tail currents measured in 2 mM extracellular K+ (open circles). Data from normally inactivating non-mutated split channels obtained in the same conditions are shown as dashed lines (Y545 Split) for comparison. Error bars represent s.e.
	Figure 9. Characterization of voltage-dependent currents elicited by co-expression of KV11.1 demi-channels carrying a Δ138–373 deletion.(a) Voltage-dependent activation of KV11.1 C-terminal and N-terminal demi-channels carrying a Δ138–373 deletion corresponding to the proximal domain located at the cytoplasmic segment, preceding helix S1 in the initial half of the voltage sensor module. Left panel, raw current traces recorded in 50 mM extracellular K+ in response to the voltage protocol shown at the top. Right panel, plot of normalized peak tail currents as a function of depolarizing voltage. A curve from non-deleted split channels obtained in the same conditions is shown as a dotted line for comparison. Error bars represent s.e. (b) Determination of voltage-dependent activation rates. Left panels, time course of current activation at 0 mV studied in 50 mM extracellular K+. Representative currents recorded in response to the illustrated voltage protocol are shown at the top. A plot of normalized peak current value versus depolarization time at 0 mV is shown at the bottom. The time necessary to attain half-maximum current magnitude is marked with a dashed arrow. Right panel, dependence of activation rates on depolarization membrane potential. Values from wild-type non-split channels (Wild type continuous), non-deleted split channels (Y545 Split) and non-split channels lacking the proximal domain at the amino terminus (Δ138–373 continuous) are also shown for comparison. (c) Voltage-dependent deactivation. Left panel, representative current traces recorded in 50 mM extracellular K+ during steps to potentials ranging from −20 to −140 mV with the voltage protocol schematized at the top. An enhanced view of the peak tail currents during the repolarization steps is shown in the inset. Right panel, plot of fast deactivation time constant for different repolarization voltages. Values from wild-type non-split channels (wild-type continuous) and non-deleted split channels (Y545 Split) are also shown for comparison.
	Figure 10. Hybrid split channels yield voltage-dependent currents.(a) Voltage-dependent activation of the KV10.1 N-terminal/KV11.1 C-terminal assembled demi-channels. Current traces as result of 500 ms depolarizations to potentials ranging between −80 and +80 mV in 20 mV intervals in both 2 and 50 mM extracellular K+ are shown at the top. The normalized I/V relationships at both potassium concentrations are shown at the bottom. (b) Voltage-dependent activation of the KV10.2 N-terminal/KV10.1 C-terminal assembled demi-channels. Representative current records in 2 mM extracellular K+ are shown. Note the much higher current level obtained with this mixture as compared with a. Normalized I/V relationships of the mixed demi-channels (n=18) as compared with KV10.1 demi-channels (n=23) and KV10.2 continuous channels (n=17) are shown at the bottom. Error bars represent s.e. (c). Acceleration of current activation (‘Cole–Moore’ shift) by holding the KV10.1 N-terminal/KV11.1 C-terminal mixed demi-channels at more depolarized potentials. The traces correspond to representative currents obtained from oocytes held at the indicated holding potentials upon stimulation to +40 mV using the protocol represented at the top.

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