Transfer of rolf S3-S4 linker to HERG eliminates activation gating but spares inactivation

Abstract

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.

Figure 1

Voltage dependence of hERG and the chimera activation. Representative whole-cell current recordings from COS-7 cells expressing (A) wild-type hERG or (B) chimeric channels. (Inset) Voltage stimulation, every 3.5 s. (C) Current density measured at the end of the prepulse versus voltage in cells expressing hERG (n = 8) or chimeric (n = 8) channels. (D) Relative tail current measured at −60 mV versus prepulse potential in cells expressing hERG (n = 8) or chimeric (n = 8) channels. hERG steady-state activation was determined by fitting hERG data with a Boltzmann function (solid line).

Figure 2

hERG and the chimera have identical inactivation. (A and C) Representative inactivating current recordings of cells expressing hERG or chimeric channels recorded with two protocols. (Inset) Three-pulse voltage protocol from holding potential −80 mV. Pulses duration: (A) first, 200 ms; second, 5 ms; third, 200 ms, every 3 s; (C) first, 1000 ms; second, 5 ms; third, 50 ms, every 3 s. (B) Steady-state current (I_ssi measured 20 ms after the beginning of the pulse) relative to the peak current (I₀) recorded during the third pulse versus voltage in cells expressing hERG (n = 8) or chimeric (n = 7) channels. Steady-state inactivation was determined by fitting the data (WT, solid circle; chimera, open circle; measured values that are not taken into account for the fit, gray circles) with a Boltzmann function (solid line). (D) Peak current (I₀) relative to maximal peak current (I_0max) estimated by fitting current traces 1 ms after depolarization and extrapolating to the beginning of the pulse (hERG, n = 6; chimera, n = 7). In the case of hERG current, I₀ is corrected from the fraction of deactivation measured during the second pulse. Steady-state inactivation was determined by fitting the data as in B.

Figure 3

hERG and the chimera have identical inactivation and recovery from inactivation kinetics. Recovery from inactivation current traces of (A) hERG and the (B) chimera. (Right) Time-expanded framed zone. (Inset) Voltage protocol, every 3 s. (C) Voltage dependence of the time constant of recovery from inactivation for hERG (n = 6) and the chimera (n = 7) and voltage dependence of the inactivation time constant for hERG (n = 9) and the chimera (n = 9).

Figure 4

Kinetic models of hERG and chimeric channels. (A, top) Representative whole-cell current recordings from COS-7 cells expressing hERG or chimeric channels. (Inset) corresponding voltage protocols. (A, bottom) Simulated currents obtained with the hERG kinetic model (B) optimized to fit hERG experimental currents. (Right) Simulated chimera currents were obtained with the chimera kinetic model (C) using the same transition rates as the hERG kinetic model. No further optimization was used or needed.

Figure 5

Effect of BeKm-1 on hERG and the chimera. Representative currents of (A) hERG and the (B) chimera in baseline conditions and after a 3-min exposure to 25 nM BeKm-1 elicited by an ascending voltage ramp (10 mV/s; every 15 s). (C) Mean fractional block (1 − (current in the presence of 25 nM BeKm-1/baseline current)) measured at various potentials during the depolarizing ramp in cells expressing hERG (n = 6) or chimeric (n = 7) channels. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (Solid triangles) Mean steady-state fractional block measured at the end of a 5-s step to −30 mV (n = 6), 0 mV (n = 7), or +40 mV (n = 7) in cells expressing hERG.

Figure 6

Effect of ketoconazole (keto) on hERG and the chimera. Representative currents of (A) hERG and (B) chimera in baseline conditions and after 3-min exposure to 5 μM keto elicited by an ascending voltage ramp (10 mV/s; every 15 s). (C) Mean fractional block (1 − (current in the presence of 5 μM keto/baseline current)) measured at various potentials during the depolarizing ramp in cells expressing hERG (n = 8) or chimeric (n = 6) channels. ^∗∗p < 0.01; ^∗∗∗p < 0.001.