Wu W et al. (2014), Stoichiometry of altered hERG1 channel gating b...

XB-ART-53393

J Gen Physiol 2014 Apr 01;1434:499-512. doi: 10.1085/jgp.201311038.

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Stoichiometry of altered hERG1 channel gating by small molecule activators.

Wu W , Sachse FB , Gardner A , Sanguinetti MC .

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Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.

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Species referenced: Xenopus
Genes referenced: kcnh2

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	Figure 1. Concatenated tetrameric hERG1 (WT4) channels have similar biophysical and pharmacological properties as channels formed by coassembly of single WT hERG1 subunits. (A) Representative WT and WT4 hERG1 channel currents recorded at the indicated Vt (−70 to 30 mV stepped in 20-mV increments). (B) The voltage dependence of WT channel gating (upright green triangles, activation; sideways green triangles, C-type inactivation) is similar to WT4 channels (upside-down black triangles, activation [n = 6]; sideways black triangles, C-type inactivation [n = 8]). Data were fitted to Boltzmann function (smooth curves) to determine V0.5act and z values (presented in Table 1). (C) Time constants (τ deact) for fast and slow components of current deactivation for WT (green triangles, n = 10) and WT4 (black triangles, n = 11) channels at the indicated return potential (Vret). (D) Effect of 10 µM PD on WT and WT4 hERG1 channel currents elicited by 4-s step to 0 mV. Itail was measured at −70 mV. (E and F) Itest-Vt relationships in the absence (black triangles) and presence of 10 µM PD (red triangles) for oocytes injected with cRNA encoding single WT subunits (E, n = 10) or WT4 channels (F, n = 7). Currents were normalized to peak Itest (at −20 mV) measured under control conditions. (G) Effect of 10 µM ICA on currents for WT and WT4 channels. (H and I) Itest-Vt relationships in the absence (black triangles) and presence of 10 µM ICA (blue triangles) for oocytes injected with cRNA encoding single WT subunits (H, n = 5) or WT4 channels (I, n = 10). Data are expressed as mean ± SEM (n = number of oocytes).
	Figure 2. Markov modeling of hERG1 tetrameric channel currents modified by drugs PD and ICA. (A) The model comprises seven states (C0,…,C4, closed states; O, open state; I, inactivated state) coupled with the indicated rate coefficients. (B) Averaged currents (n = 4) from oocytes expressing WT4 tetramers under control conditions in response to voltage steps to the indicated Vt. (C) Simulated currents for WT4 tetramers under control conditions. (D and E) Averaged currents measured from the same oocytes as in B, after application of 30 µM PD (D), and corresponding simulated currents (E). (F and G) Averaged currents (n = 6) measured after application of 30 µM ICA (F) and corresponding simulated currents (G). Currents shown in B, D, and F were not leak subtracted.
	Figure 3. Biophysical properties of concatenated LEn/WT4−n tetrameric hERG1 channels. (A) Itest-Vt relationships. (B) Voltage dependence of activation. (C) Fully activated Itail-Vret relationships. (D) Voltage dependence of inactivation. Legend refers to all panels. Data are expressed as mean ± SEM (n = 5–12; values for V0.5 and z for activation and inactivation are presented in Table 1; n = number of oocytes).
	Figure 4. PD-induced enhancement of hERG1 current indicates cooperative subunit interactions. (A) Representative current traces recorded at 0 mV for concatenated tetramers containing zero to three WT subunits together with one to four L646E subunits, before and after 10 µM PD. (B) Effect of 10 µM PD on voltage dependence of channel activation. Itail was measured at −70 mV after pulses to the indicated Vt and normalized to Itail-max under control conditions for each channel type (n = 7–15). The symbol legend between B and E refers to data plotted in B and D–F. Control Itail-Vt relationship (black pentagons) overlaps data for LE4 channels and represents the mean of all channel types. The V0.5act and z values obtained from fitting Itail-Vt relationships to a Boltzmann function (smooth curves) before and after 10 µM PD are presented in Table 1. (C) Pulse protocol (top) used to measure fully activated Itail-Vret relationship and representative currents for WT4 channels under control conditions and after 10 µM PD as indicated. (D) Effect of 10 µM PD on Itail-Vret relationships (n = 4–9) normalized to Itail at −120 mV under control conditions for LEn/WT4−n tetramers (n = 0–4). (E) [PD]-response relationships for fold increase in Itail-max at −70 mV for different tetramers according to symbol legend shown between B and E. Data were fitted with a logistic equation to determine EC50 values and Hill coefficient, nH (see Fig. 5). (F) Plot of ΔG versus number of WT subunits contained in a concatenated tetramer. ΔG was equal to −RTlnKeq, where Keq was defined as Po/(1 − Po) at maximal effect of PD and assuming maximum Po in the absence of drug to be 0.5. Linear regression analysis was used to fit calculated data (colored symbols; solid line: y = −0.39x + 0.058; R2 = 0.96) and the relationships predicted for independent subunit transitions (▾) and cooperative subunit interactions, models 1 (▴) and 2 (•). Data are expressed as mean ± SEM (n = number of oocytes).
	Figure 5. The concentration response relationship for PD-induced increase in Itail is not dependent on the number of WT subunits in a concatenated tetramer. (A) Plot of EC50 as a function of the number of WT subunits in a tetramer. Line represents linear fit to data: y = 0.64x + 1.22 (adjusted R2 = 0.099). (B) Plot of nH as a function of number of WT subunits in a tetramer. Line represents linear fit to data: y = −0.015x + 1.24 (adjusted R2 = −0.473). In both panels, the open circle represents LE1/WT1/LE1/WT1 channels; the open square represents WT3/LE1 channels.
	Figure 6. Positioning of a single LE mutant subunit in a concatenated hERG1 tetramer does not affect biophysical properties or response to PD. (A) Representative current traces for LE1/WT3 and WT3/LE1 hERG1 channels. Pulses were applied to Vt of −70 to 30 mV in 20-mV increments. Vh was −80 mV and Vret was −70 mV. (B) Normalized Itail-Vt relationships before and after 10 µM PD. Symbol legend is the same as for C. For LE1/WT3 hERG1 channels: V0.5act = −32.1 ± 1.3 mV, z = 4.13 ± 0.14 mV (control); V0.5act = −30.1 ± 1.6 mV, z = 4.20 ± 0.07 mV (PD; n = 11). For WT3/LE1 hERG1 channels: V0.5act = −29.1 ± 1.5 mV, z = 3.36 ± 0.21 mV (control); V0.5act = −27.1 ± 1.3 mV, z = 3.44 ± 0.16 mV (PD; n = 4). (C) Normalized fully activated Itail-Vret relationships determined before and after 10 µM PD. (D) [PD]-response relationships for LE1/WT3 hERG1 channels (EC50 = 4.6 µM, nH = 1.1; n = 6–9) and WT3/LE1 hERG channels (EC50 = 4.8 µM, nH = 1.4; n = 4). Data are expressed as mean ± SEM (n = number of oocytes).
	Figure 7. Biophysical properties of concatenated FLn/WT4−n tetrameric hERG1 channels. (A) Representative current traces for concatenated tetramers containing zero to three WT subunits together with one to four F557L subunits. Pulses were applied to Vt of −70 to 30 mV in 20-mV increments. Vh was −80 mV and Vret was −70 mV. (B) Itest-Vt relationships for FLn/WT4−n tetrameric hERG1 channels. (C) Voltage dependence of activation. (D) Fully activated Itail-Vret relationships. (E) Voltage dependence of inactivation. The symbol legend refers to B–E. Data are expressed as mean ± SEM (n = 3–12). Values for V0.5 and z for activation and inactivation are presented in Table S2.
	Figure 8. Concentration- and voltage-dependent effects of ICA on Itest. (A) Concentration-dependent increase in Itest measured at 20 mV by ICA for WT4 and FL1/WT1/FL2 hERG1 concatenated channels. Data were fitted with a logistic equation (smooth curves). For WT4 channels: EC50 = 9.1 µM, nH = 1.7 (n = 6–9); for FL1/WT1/FL2 channels: EC50 = 11.8 µM, nH = 2.0 (n = 6–10). (B) Fold increase in Itest by 30 µM ICA as a function of Vt (n = 3–8). Data are expressed as mean ± SEM (n = number of oocytes).
	Figure 9. Attenuation of hERG1 inactivation by ICA exhibits positive cooperativity. (A) Representative current traces under control conditions and after 10 µM ICA for tetramers containing the indicated number and orientation of WT and F557L subunits. (B) Normalized Itest-Vt relationships for concatenated tetramers measured in the presence of 30 µM ICA (n = 4–10). Currents were normalized to the peak Itest under control conditions for each channel type. Symbol legends refer to data plotted in B, C, and G. (C) Correlation between log fold increase in Itest at 20 mV induced by 30 µM ICA and the number of WT subunits (i.e., number of functional ICA binding sites) in a concatenated tetramer. Data were fitted with a linear function: y = 0.36x + 0.1 (R2 = 0.94). (D) Fully activated Itail-Vret relationship for FL1/WT3 and FL1/WT1/FL2 tetramers before and after 30 µM ICA (n = 4). Currents were normalized to control Itail at −120 mV. (E) Voltage dependence of inactivation for FL1/WT3 and FL1/WT1/FL2 tetramers before and after 30 µM ICA (n = 4). V0.5inact and z values are presented in Table 2. (F) Effect of 30 µM ICA on the fully activated Itail-Vret relationships for WT4 channel currents recorded from oocytes bathed in a solution containing 20 mM KCl (n = 4). Data were normalized to Itail measured at −120 mV under control conditions. (G) Plot of calculated energy values, ΔΔG = \|zFV0.5inact(30μM ICA)−zFV0.5inact(control)\|, versus number of WT subunits contained in a concatenated tetramer compared with the relationships predicted for independent subunit transitions (▾) and cooperative subunit interactions, models 1 (▴) and 2 (•). Data are expressed as mean ± SEM (n = number of oocytes). Standard errors in zFV0.5inact were calculated as described previously (Yifrach and MacKinnon, 2002).

References [+] :

Abbruzzese, Modification of hERG1 channel gating by Cd2+. 2010, Pubmed, Xenbase