Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and eag potassium channels

Abstract

Unintended block of HERG K+ channels is a side effect of many common medications and is the most common cause of acquired long QT syndrome associated with increased risk of life-threatening arrhythmias. The molecular mechanism of high-affinity HERG block by structurally diverse compounds has been attributed to pi-stacking and cation-pi interactions of a drug (e.g., cisapride) with specific aromatic amino acid residues (Tyr-652 and Phe-656) in the S6 alpha-helical domain that face the central cavity of the channel. It also has been proposed that strong C-type inactivation of HERG facilitates or is the primary determinant of high-affinity drug binding. The structurally related, but noninactivating eag channel is insensitive to HERG blockers unless inactivation is induced by specific amino acid mutations [Ficker, E., Jarolimek, W. & Brown, A. M. (2001) Mol. Pharmacol. 60, 1343-1348]. Here we examine the relative importance of inactivation vs. positioning of S6 aromatic residues in determining sensitivity of HERG and eag channels to block by cisapride. The repositioning of Tyr-652 or Phe-656 along the S6 alpha-helical domain of HERG reduced sensitivity of channels to block by cisapride. Moreover, independent of inactivation, repositioning of the equivalent aromatic residues in Drosophila eag channels induced sensitivity to block by cisapride. These findings suggest that positioning of S6 aromatic residues relative to the central cavity of the channel, not inactivation per se determines drug block of HERG or eag channels.

Fig 1.

Sequences of S6 domain for HERG and Drosophila eag channels and the location of introduced point mutations. (a) Sequence of wt and mutant HERG S6 domains with Tyr-652 and Phe-656 highlighted in bold type. (b) Sequence of wt and mutant eag S6 domains with Tyr-451 and Phe-455 highlighted in bold type. (c) Diagram showing position of Phe residue in wt and repositioning in mutant HERG channels (*, HERG/F-up; **, HERG/F-down).

Fig 2.

Effect of repositioning S6 aromatic residues in HERG on biophysical properties and sensitivity to bock by cisapride. (a) Voltage pulse protocol and endogenous currents in an uninjected oocyte. (b) Block of wt HERG channel current by 30 nM, 100 nM, and 0.3 μM cisapride. (c and d) HERG/Y-up and HERG/F-down channels are only partially blocked by 10 μM cisapride. Note that HERG/F-down channel current is time-independent. (e) Effect of 0.3, 1, and 3 μM cisapride on HERG/F-up channel current. (f) Concentration–effect relationship for block of wt (▪), Y-up (•), F-down (▾), and F-up (▴) HERG channels by cisapride. The IC₅₀ was 0.102 ± 0.001 μM and 1.60 ± 0.11 μM for wt HERG and HERG/Y-up (n = 4). (g) Voltage dependence of channel availability for wt HERG and HERG/F-up channels, determined with a triple pulse protocol. Data were fitted with a Boltzmann function (curve) to estimate one-half point (V_1/2) and slope factor (k): wt-HERG (▪, V_1/2 = −81.2 ± 1.4 mV, k = 25.0 ± 1.3 mV, n = 7) and HERG/F-up (▴, V_1/2 = −85.3 ± 1.3 mV, k = 21.5 ± 1.2 mV, n = 8).

Fig 3.

Effect of cisapride on wt and mutant eag channel currents. (a–g) wt and mutant eag currents recorded during a 2.5-s pulse to 0 mV before and after steady-state effects of 3 μM cisapride (*). (h) Concentration-effect relationships for wt and mutant eag channels. The IC₅₀ was 11.9 ± 1.2 μM for wt eag, 3.7 ± 0.3 μM for eag/Y-up, 0.28 ± 0.05 μM for eag/Y-down, 4.6 ± 0.4 μM for eag/F-up, 1.3 ± 0.1 μM for eag/F-down, and 18.9 ± 0.3 μM for eag/YF-down (n = 4–6). The 30 μM cisapride blocked eag/YF-up current by 11% ± 3% (n = 5).

Fig 4.

Voltage dependence for inactivation of mutant eag channels. (a–f) Voltage pulse protocol and currents used to assess inactivation properties of the indicated mutant channel. Prepulses (V_pre) were applied from −180 to −90 mV for eag/F-down (a) and from −140 to +10 mV for all other mutant channels. The test pulse (V_t) was applied to 0 or +20 mV as indicated. (g) Voltage dependence of channel availability of eag mutant channels. Peak or extrapolated peak currents recorded at V_t were normalized and plotted vs. V_pre. Data were fitted with a Boltzmann function to obtain the one-half point (V_1/2) and slope factor (k) for the relationship. The one-half point and slope factor were −113.6 ± 0.3 mV and 11.7 ± 0.3 mV for eag/F-down (□); −68.6 ± 0.3 mV, 8.9 ± 0.3 mV for eag/Y-down (•); −57.4.3 ± 0.3 mV, 9.3 ± 0.3 mV for eag/A477G/Y-down (▴); and −45.1 ± 0.2 mV, 11.7 ± 0.2 mV for eag/A478G (◃). eag/A478G/Y-down (♦) and eag/A477G (▾) channels did not inactivate within the test range (n = 7–11).

Fig 5.

Potency for block of mutant eag channels is not correlated with extent of inactivation. (a) The IC₅₀ for block by cisapride was 0.28 ± 0.05 μM for eag/Y-down, 0.41 ± 0.04 μM for eag/A477G/Y-down, 0.33 ± 0.03 μM for A478G/Y-down, 16.8 ± 1.5 μM for eag/A477G, and 1.5 ± 0.2 μM for eag/A478G (n = 4–6). (b) IC₅₀ for block by cisapride plotted as a function of the percentage of channels inactivated at 0 mV.