Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Abstract

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.

FIGURE 1.

An Ala scan of the hERG1 pore helix reveals residues critical for inactivation. A, sequence alignments of hERG and K⁺ channels whose crystal structure has been solved. The S5 domain sequences are aligned by a Glu (E) that defines the extracellular end of the S5 (or outer helix) of the Kv1.2–2.1 chimera (37), KvAP (38), KcsA (39), and MthK (40) crystal structures. hERG1 residues in bold type represent key positions that perturb inactivation gating when mutated. The black bar demarcates the residues mutated in the Ala scan. For simplicity, the 32 amino acids comprising the S5-P linker turret are represented as a hatched box. B, WT and T618A hERG1 ionic currents recorded in solutions containing [K⁺]_o = 4 mm (left panel) and 96 mm (right panel). Currents were elicited by a 2-s depolarization to +40 mV, followed by a voltage step to potentials between −140 mV and +40 mV in 10-mV increments from a HP = −80 mV. Displayed currents correspond to the time period shown as a dotted box surrounding the voltage protocol. The arrows indicate zero current. C, I-inact-V relationships for WT (■),T618A (●), T623A (△), V625A (○) and F627A (<) using a two-pulse protocol in B in 96 mm K. D, bar graph representing mean ± S.E. for ΔΔG₀ inactivation (kcal-mol⁻¹) for residues in the pore-helix scan. The dashed line represents the arbitrary designation of a mutation-induced perturbation in inactivation (ΔΔG₀ ≥ (1 kcal-mol⁻¹). n = 6–10 cells. NE, no functional expression. Inactivation parameters for WT and all mutant constructs are listed in supplemental Table 1.

FIGURE 2.

Characterization of hERG1 mutants that did not display ionic current. The inability to detect ionic current from six Ala mutants suggests a disruption in protein targeting to the cell surface or defective ion conduction. To distinguish between these two possibilities, we measured gating currents and cell surface expression by Western blot analysis. A, representative gating current traces elicited by 300-ms step depolarization to +40 mV from HP −110 mV using a p/-8 leak subtraction protocol. T611A, A614V, Y616A, and F617A hERG1 channels failed to generate detectable gating currents. G626A and G628A hERG1 channels generated small but detectable gating currents qualitatively similar to WT hERG1a. The X-Y legend represents 100 ms and 0.2 μA, respectively. B, to further characterize cell surface targeting, we used Western blot analysis to compare the expression patterns of the immature core-glycosylated (cg) and mature fully glycosylated (fg) mature protein as described (41). For the four mutants that did not generate gating current, the fully glycosylated band was absent, confirming that these mutations disrupted cell surface expression. By contrast, the fully glycosylated protein was detected for the two mutants that generated gating current, confirming that these mutant subunits were targeted to the cell surface but failed to conduct ions.

FIGURE 3.

The effects of selectivity filter mutations on hERG1 inactivation. A, selectivity filter residues identified in the pore helix Ala scan that perturbed inactivation were subjected to additional mutagenesis to determine the side chain requirements for normal inactivation gating. The bar graph represents mean ± S.E. for ΔΔG₀ inactivation (kcal-mol⁻¹). n = 4–8 cells. NE, no functional expression. B, homology model of the hERG1 pore domain, based on the Kv1.2 crystal structure, highlighting the residues that perturbed inactivation gating. The position of the Trp-568 side chain is shown in transparent gray.

FIGURE 4.

Thr-618 mutations exert opposite effects on hERG1 inactivation gating, and T618V alters gating charge associated with inactivation. A, ΔΔG₀ values for inactivation of Thr-618 mutants (n = 4–8 cells). B, conductance-voltage relationships for the T618A and T618V hERG1 mutants. V_1/2 and z were −22.8 ± mV and 3.0 ± 0.1 mV for T618A and −20.0 ± mV and 4.0 ± 0.1 mV for T618V (n = 6–9 cells). C, a single trace showing the T618V gating current (I-gating) elicited a 0.3-s voltage step to +80 mV, a 10-ms interpulse to 130 mV, and return to +80 mV (see “Experimental Procedures”). Currents in the right panel represent an expanded view of the area marked by the dotted box. The inset shows the integral of gating currents measured during the third voltage step to +80 mV. The WT hERG1 gating current is shown below. D, I-Inact and Q-Inact relationships elicited by the triple-pulse voltage protocol in [K⁺]_o = 4 mm and 120 mm symmetric TEA⁺ solutions, respectively. T618V caused a rightward shift in the Q- and I-Inact relationship compared with the WT.

FIGURE 5.

A point mutation in the S5 domain abolishes hERG1 inactivation and alters selectivity. A and B, W568L currents (left panel) elicited by the indicated voltage protocol and I-V relationships (right panel) using current magnitude as measured at the diagonal arrow. The horizontal arrow marks the position of zero current. The I-V relationship for WT hERG1 is shown as ○. C, ΔΔG₀ values of inactivation for Trp-568 mutants. NE, no expression.

FIGURE 6.

Other Trp-568 mutations alter hERG1 inactivation gating. A and B, representative currents for W568I and W568F hERG1 channels elicited by step depolarizations in 4 mm or 96 mm external K⁺ using the indicated voltage protocols. C, I-V relationships for W568I and W568F hERG1 as measured from B. Tail currents were normalized to peak tail current at −160 mV (n = 5 cells). D, inactivation-voltage relationship for W568F hERG1 measured with a two-pulse voltage protocol in 96 mm external K⁺. WT data is plotted from Fig. 2B. The V_1/2 and z for W568F hERG1 were −94.1 ± 2 mV and 0.7 ± 0.03 mV (compared with −45.7 mV and 0.8 mV for WT hERG1). E, description of the electrophysiological phenotype of double mutant 568–618 hERG1 mutants. NE, no functional expression.

FIGURE 7.

Effects of introducing Thr at the hERG 618-equivalent position in Kv1.5 and an atomic model of Kv1.2 channels. A, introducing Thr at the hERG 618-equivalent position in Kv1.5 channels (A473T) markedly impacts inactivation. A473T Kv1.5 currents elicited by repetitive voltage steps to +40 mV from a HP = −80 mV and 15-s interpulse duration. B, comparison of the time course of reduction in peak current versus time for WT and A473T Kv1.5 channels. Each data point represents the mean ± S.E. of peak current magnitude normalized to peak current following the initial depolarization (n = 4 cells). C, MD simulations using a full atom representation of the Kv1.2 pore domain embedded in lipid. Interaction energies between residue 368 and individual side chains of the S5 domain residues 340–346 are calculated from the last 4 ns of MD simulations in a model of the Kv1.2 tetramer. Ala-368-Phe-342 and Ala-368-Ser-343 van der Waal interactions are significantly more favorable than interactions between Ala-368 and the flanking S5 residues (**, p < 0.00001). The A368T mutation does not alter these interactions. However, A368T markedly increases the electrostatic interaction with Ser-343 compared with the WT (*, p < 0.0001). Ala-368 and Ser-343 correspond to Thr-618 and Trp-568 in hERG1, respectively. Data represent mean ± S.D.