Dhillon MS , Cockcroft CJ , Munsey T , Smith KJ , Powell AJ , Carter P , Wrighton DC , Rong HL , Yusaf SP , Sivaprasadarao A .

Biotechnology and Biological Sciences Research Council

	Figure 1. Sequence alignment of Kv1.2 and hERG showing sites of substitutions.Potential transmembrane regions (highlighted in yellow) and other functional elements (S4–S5 linker and pore-helix) are labelled. Arrows indicate sites where different chimaeras were joined: Black, S1–S6; red, S4/S5–S6; blue, P-S6.
	Figure 2. Functional analysis of hERG-Kv1.2 chimaeras.(a) Schematic of constructs showing hERG segments (gray) substituted into Kv1.2 (black and white) (b) Representative current families recorded from oocytes injected with cRNA corresponding to the constructs in (a). Currents for the chimaeric channels were recorded using the hERG voltage step protocol (−80†mV to +40†mV in 10†mV intervals, followed by a step to −50†mV; interpulse interval was 20†s). Currents through KV1.2 were recorded at various step potentials (−80†mV to +80†mV) delivered in 10†mV increments from a holding potential of −80†mV; interpulse interval was 10†s. Current traces for hERG and the S1–S6 chimaera are shown in colour (hERG: −80 to 0†mV, blue; +10†mV, green; +20 to +40†mV, red; and S1–S6 chimaera: −80 to −10†mV, blue; 0†mV, green; +10 to +40†mV, red). Also shown is the scaled version of tail currents for the S1–S6 chimaera. (c) Schematic of the protocol used to record currents through hERG and the chimaeras. (d) Plots of voltage against steady-state currents (mean ± SEM) recorded at the time point indicated by a black arrow in C. * indicates currents through hERG are significantly greater than the S1–S6 chimaera (p < 0.05). (e) Plots of voltage against peak tail currents (mean ± SEM) recorded at the beginning of the −50†mV pulse (corresponding to gray arrow in c). The lines joining the mean currents represent Boltzmann distribution. The V1/2 value of the S1–S6 chimaera (−10.78 ± 3.55†mV, n = 13) is not significantly different (p > 0.05) from that of hERG (−18.03 ± 0.90†mV, n = 20). However, slope (k) values are significantly different (p < 0.05) between hERG (6.99 ± 0.22, n = 20) and the S1–S6 chimaera (12.94 ± 1.66, n = 13). (f) Normalised plot of data from (e).
	Figure 3. Comparison of the activation kinetics of hERG and the S1–S6 chimaera.(a) Comparison of representative channel activation current traces between hERG (black traces) and the S1–S6 (grey traces) chimaeras at the indicated voltage steps. (b–c) Comparison of the voltage dependence of fast (b) and slow (c) activation time constants calculated from the activation current traces, as described in methods (chimaera, n = 8; hERG, n = 5). The fast as well as slow activation kinetics of the S1–S6 chimaera are significantly faster than those for hERG (p < 0.05) at all potentials examined.
	Figure 4. Comparison of the deactivation kinetics of hERG and the S1–S6 chimaera.(a) Voltage protocol used to measure deactivation kinetics. (b) Representative deactivation current traces of hERG and the S1–S6 chimaera at various voltage potentials following the +40†mV pulse. (c–d) Comparison of the voltage dependence of fast (b) and slow (c) deactivation time constants calculated from deactivation current traces, as described in methods (chimaera, n = 8; hERG, n = 5). The fast deactivation kinetics of the S1–S6 chimaera are significantly faster than those for hERG (p < 0.05). (e) Normalised current-voltage relationships of peak fully activated currents (n = 7). Erev values were calculated by fitting the linear section of the curve and calculating the x-intercept (not shown). Erev values were ≈−90†mV.
	Figure 5. Comparison of recovery from inactivation of hERG and the S1–S6 chimaera.(a) The three stage voltage protocol used to measure recovery from inactivation. Voltage was first stepped from −80†mV to + 35†mV to fully inactivate the channels, then stepped from −145 to +35†mV, in 10†mV increments, followed by a third +35†mV step, and then a final −145†mV step to relieve inactivation. (b) Representative current traces for hERG and the chimaera using the protocol shown in (a). The scaled-up traces show currents during the third +35†mV step; they represent the currents recovered from inactivation during the preceding voltage steps. (c) Voltage-dependence of the recovery from steady-state inactivation. The normalised peak recovered currents were plotted against voltage and fitted with a Boltzmann function. There was a significant (p < 0.05,) rightward shift in the V1/2 value of ≈40†mV for the S1–S6 chimaera (−17.09 ± 2.37, n = 12) compared to hERG (−60.47 ± 6.20†mV). (d) Voltage dependence of the time to reach peak current (IMax); the S1–S6 chimaera takes significantly (p < 0.05) longer time compared with hERG to reach peak current over the voltage range examined.
	Figure 6. The S1–S6 chimaera is inhibited by dofetilide, the classical hERG pore blocker.(a) A three step voltage protocol used to determine inhibition by dofetilide. (b–c) Representative current traces for hERG (b) and the S1–S6 chimaera (c) before (black trace) and during (light gray trace) application of 10†μM dofetilide and after washout of the drug (dark gray). (d) Dofetilide concentration-response relationship for hERG and the S1–S6 chimaera. IC50 value for inhibition by dofetilide for the S1–S6 chimaera (39.54 ± 8.68†nM) was not significantly different from that for hERG (19.32 ± 9.6†nM) (p > 0.05; n = 4).
	Figure 7. Expression and purification of the S1–S6 channel protein in Pichia pastoris.(a) Western blots of Kv1.2 and the S1–S6 chimaera. Methanol induced cells pelleted from 1†ml culture (at a density of O.D. 8.0) were extracted into 200†μl of SDS sample buffer using glass beads and serial dilutions of the extract were subjected to western blotting using anti-His antibodies (b) Purification of the S1–S6 chimaera by Talon metal affinity chromatography. 45†mg of solubilised membrane proteins (input) were subjected to purification as described in methods. E1–E7 represent fractions eluted with 500†mM imidazole. 5†μl of each fraction were subjected to SDS-PAGE and Coomassie blue staining. (c) Western blotting of diluted fractions from the affinity purification. The input (lane 1) and flowthrough were loaded at a dilution of 1:50, whilst all the other samples were loaded at 1:10 dilution.

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