Mitcheson JS et al. (2000), Trapping of a methanesulfonanilide by closure o...

XB-ART-11431

J Gen Physiol 2000 Mar 01;1153:229-40.

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Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate.

Mitcheson JS , Chen J , Sanguinetti MC .

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Deactivation of voltage-gated potassium (K(+)) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC(50) = 32 nM) of HERG K(+) channels. This bulky compound (7 x 20 A) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 microM MK-499 to occur with time constants of 10.5 and 52.7 s at -160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K(+) channel.

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Species referenced: Xenopus
Genes referenced: kcnh2 tbx2

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	Figure 1. Electrophysiological properties of D540K HERG channels expressed in Xenopus oocytes. (A) Representative whole cell recordings of D540K HERG currents elicited by 2-s depolarizing test pulses from −60 to +20 mV in 20-mV increments at a stimulation frequency of 0.1 Hz. (B) D540K HERG currents from the same cell as A, elicited by 5-s hyperpolarizing test pulses from −100 to −160 mV in 20-mV increments at a stimulation frequency of 0.09 Hz. The holding potential was −90 mV, tail currents were recorded at −70 mV, and dotted lines indicate zero current level. (C) Inactivation-removed mutant HERG (D540K:G628C:S631C) currents activated by pulses ranging from −50 to +50 mV (top) and −100 to −160 mV (bottom). (D) Steady state I-V relationship for D540K HERG (n = 17 cells).
	Figure 2. Two components of inward current elicited by hyperpolarization. (A) Membrane potential was stepped from a holding potential of −90 to +40 mV for 500 ms to maximally activate depolarization-dependent current, and then stepped to −160 mV for 5 s. (B) Currents elicited by voltage protocol in A. Sampling frequency was changed from 1 to 5 kHz just before hyperpolarization. The transient current component represents rapid recovery from inactivation followed by deactivation, whereas the slow current represents channel reactivation. (C) Expanded view of current after hyperpolarization to −160 mV. Current deactivation was fit with an exponential function (solid line) and extrapolated back to time zero to correct for deactivation during recovery from inactivation.
	Figure 3. Comparison of WT and D540K HERG single channel currents. Currents were recorded in cell-attached patches of Xenopus oocyte membrane. (A) Voltage protocol used to elicit single channel currents. From a holding potential of −80 mV, membrane potential was stepped to +40 mV for 500 ms, followed by a 5-s step to either −80, −140, or −160 mV. Representative WT HERG (B) and D540K HERG (C) single channel currents were recorded at the indicated membrane potentials. Dotted lines indicate zero current level. (D) Mean single channel I-V relationships for WT (▿, n = 5) and D540K (▴, n = 12) HERG. Standard error bars were smaller than the symbols. WT HERG data between −110 and −60 mV were taken from Zou et al. 1997.
	Figure 4. Ensemble average of D540K HERG single channel currents. (A) Voltage protocol used to elicit single channel currents. (B) Representative currents recorded during five different pulses, showing that channel activity increased throughout the 5-s pulse to −160 mV. This patch contained at least six single channels, with an average amplitude of 2.15 pA corresponding to a conductance of 22 pS. (C) Ensemble average of 17 current traces, including those shown in B.
	Figure 6. Concentration-dependent block of HERG by MK-499. (A) Structure of MK-499 showing pKa values for nitrogen groups. (B) Examples of WT (i) and D540K HERG (ii) currents before and after exposure of oocytes to solutions containing various concentrations of MK-499. From a holding potential of −90 mV, currents were recorded during 5-s pulses to 0 mV; tail currents were recorded at −70 mV. (C) Concentration-response relationships for block of WT and D540K HERG current in oocytes. Steady state peak current amplitudes at 0 mV for each [MK-499] were normalized to control. Mean values were plotted against [MK-499] and fitted with the Hill equation. Mean IC50s were 32 ± 4 nM (Hill coefficient = 1; n = 4) for WT HERG and 104 ± 8 nM (Hill coefficient = 1.8; n = 5–7) for D540K HERG.
	Figure 5. Open probability of D540K HERG channels is voltage dependent. Representative gap-free recordings of single channel activity from a single patch recorded at −80 mV (A) and −160 mV (B). Open probability was calculated at 1-s intervals and plotted against time for −80 mV (C) and −160 mV (D). (E) Bar graph of Po-voltage relationship for D540K HERG single channel currents (n = 3–6), determined from idealized traces of gap-free current recordings with a minimum duration of 2 min.
	Figure 7. WT HERG channels do not significantly recover from MK-499 block. (A, top) Voltage pulse protocol, (bottom) WT HERG currents. Xenopus oocyte expressing WT HERG channels was continuously stimulated at a frequency of 0.1 Hz with 5-s voltage steps to 0 mV from a holding potential of −90 mV. The oocyte was pulsed until currents reached steady state (a) before switching to a solution containing 2 μM MK-499. When block of WT HERG currents was more than 85% (b), repetitive 5-s hyperpolarizing voltage steps to −160 mV were applied (45 total) at a frequency of 0.05 Hz, in the continuous presence of MK-499. c and d are the first and last hyperpolarizing pulses, respectively. Tail currents were induced by steps to −70 mV. The extent of recovery was determined with a 5-s depolarizing pulse to 0 mV (e). (B) Peak current magnitudes for experiment in A. Each point represents current elicited by a single voltage pulse. Arrow designates time at which MK-499 was administered.
	Figure 9. D540A HERG channels do not significantly recover from MK-499 block. The same protocol as described in Fig. 7 was used. (A, top) Voltage pulses, (bottom) representative D540A HERG current traces. Note that D540A HERG channels do not open with hyperpolarization. a and b are currents elicited before and after block by MK-499, respectively. c and d are currents elicited by the first and last of 50 hyperpolarizing pulses to −160 mV. e is current in response to a depolarizing voltage step applied immediately after the final hyperpolarizing pulse. (B) Peak current magnitudes for the experiment in A. Each point represents current elicited by a single voltage pulse. Arrow designates time at which MK-499 was administered.
	Figure 8. Untrapping of MK-499 from D540K HERG channels. Recovery from MK-499 block of D540K HERG was investigated using the same protocol described in Fig. 7. (A) Voltage protocol and representative current traces. a and b are currents elicited before and after block by MK-499, respectively. c and d are currents elicited by the first and last of 38 hyperpolarizing pulses. e is current in response to the first depolarizing voltage step after the hyperpolarizations. (B) Peak current magnitudes for experiment in A. Each point represents current elicited by a single voltage pulse. Arrow designates time at which MK-499 was administered.
	Figure 10. Rate of D540K HERG recovery from block is voltage dependent. Representative current traces elicited by 2-min hyperpolarizing voltage steps to −120 mV (A) or −160 mV (B). Control currents were elicited once outward currents stimulated with 5-s pulses to 0 mV had reached steady state. After the addition of 2 μM MK-499, the oocyte was pulsed repetitively to 0 mV to achieve >85% block. Then the test pulse to −120 was repeated. Block was reestablished before recording current at −160 mV. (C) Drug sensitive current, Id-s was calculated by subtracting the currents in the presence of MK-499 from the control currents, Ic, at each potential. Id-s was normalized to control current (Id-s/Ic, dot plot) for each potential and fitted with exponential functions (solid lines) to obtain time constants for recovery from MK-499 block. Id-s/Ic at −120 mV was fitted with a single exponential function (τ = 109 s). Id-s/Ic at −160 mV was fitted with a biexponential function (τf = 10.5 s, τs = 52.7 s).

References [+] :

Abbott, MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. 1999, Pubmed, Xenbase