Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Abstract

Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

Keywords: Kv channel, S1-S2 linker, human beta-defensin 2, electrostatic interaction, gating modifier; defensin; immunology; ligand-binding protein; toxin; voltage-dependent anion channel (VDAC).

FIGURE 1.

Kv1.3 channel activation modified by hBD2. A and B, voltage-dependent inhibition of Kv1.3 channel currents by hBD2. 69.8 ± 2.4% and 40.9 ± 0.8% of Kv1.3 channel currents were inhibited by 10 pm hBD2 at −30 and 0 mV, respectively. The voltage protocol is placed at the top. C, activation G-V curves of the Kv1.3 channels modified by hBD2. Peak current amplitude during each activation step was used to generate G-V curves. Activation G-V curves of the Kv1.3 channels were plotted with the Boltzmann equation. The V₅₀ values were −32.75 ± 1.01 and −22.27 ± 0.60 mV in the absence and presence of 10 pm hBD2, respectively. D, activation time constants of Kv1.3 channels are plotted as a function of voltages before (black) and after (red) applying 10 pm hBD2.

FIGURE 2.

Effects of extracellular domains of Kv channels on hBD2 binding. A, sequence alignment between the human Kv1.2 and Kv1.3 channels. The S1-S2, S3-S4, and S5-S6 linkers of the Kv1.2 and Kv1.3 channels are colored with light cyan and pink, respectively. The secondary structure features are indicated above the sequences. Conserved basic residues in the S4 segment are highlighted in blue. The chimeric Kv1.2 channels were generated based on differences in the three colored extracellular domains between the Kv1.2 and Kv1.3 channels. B–D, representative current traces of the chimeras inhibited by hBD2. 51.7 ± 2.7% of the S5-S6 linker chimera currents blocked by 10 nm hBD2 (B), 9.2 ± 1.9% of the S3-S4 linker chimera currents blocked by 1000 nm hBD2 (C), and 54.2 ± 0.9% of the S1-S2 linker chimera currents blocked by 1 nm hBD2 (D). E, concentration dependence of the hBD2 inhibition of the Kv1.2 channel chimeras and the Kv1.3 channel. F, abridged general view of the Kv1.2 channel chimeras and the IC₅₀ values for the hBD2 interactions with different potassium channels. G, co-immunoprecipitation of Kv1.3, Kv1.2, and the S1-S2 linker chimera channels with hBD2.

FIGURE 3.

Effects of the extracellular domains of Kv channels on the capability of hBD2 modifying channel activation. A–D, the G-V curves from the peak currents were plotted for the Kv1.2 (A), S1-S2 linker chimera (B), the S3-S4 linker chimera (C), and the S5-S6 linker chimera (D) in the absence and presence of hBD2. A significant G-V curve shift was only observed in the S1-S2 linker chimeric channel. E, an abridged general view of the Kv1.2 chimeras and the detailed V₅₀ values before and after hBD2 interacts with different channels. The ΔV₅₀ = V₅₀(+hBD2) − V₅₀(−hBD2).

FIGURE 4.

The positive charges in S4 influencing hBD2 to modify activation of the Kv1.3 channel. A, sequence alignment of the S1-S2 and S3-S4 linkers between the human Kv1.2 and Kv1.3 channels. The mutant residues in the human Kv1.3 channels were marked. B, the positive charges in S4 influencing hBD2 to modify activation of the Kv1.3 channel. The G-V curves from the peak currents were plotted for the Kv1.3-A309R channel, the Kv1.3-R312H/R315H channel, and the Kv1.3-R312S/R315S channel in the absence and presence of hBD2. The V₅₀ values were −24.67 ± 1.71 and 6.57 ± 1.55 mV for the Kv1.3-A360R channel, 7.09 ± 0.77 and 11.45 ± 0.86 mV for the Kv1.3-R312H/R315H channel, and 4.89 ± 0.60 and 7.10 ± 0.77 mV for the Kv1.3-R312S/R315S channel before and after interacting with hBD2, respectively. C, co-immunoprecipitation of Kv1.3 and three mutants in the S1-S2 linker. The ranking of the changes in the ΔV₅₀ values was as follows: Asp-209 > Phe-207 > Asp-220. The binding affinity with hBD2 was: D209A < F207A < D220A, which indicated that loss of hBD2 in the S1-S2 mutants could simply be caused by the loss of hBD2 binding. D, co-immunoprecipitation of Kv1.3 and three mutants in the S4 segment. The Kv1.3-R312H/R315H and Kv1.3-R312S/R315S channels, which had changes in the S4 charge, showed a loss of shift in V₅₀, but the channels could still interact with hBD2. The amount of interaction was the same as the Kv1.3-A309R channel. E, the key residues in the S1-S2 linker of the Kv1.3 channel for hBD2 binding. The S1-S2 linker structure of the Kv1.3 channel was modeled by using the Kv1.2 structure as a template (Protein Data Bank code 3LUT). All nine critical residues were located on the N and C termini of the S1-S2 linker. The polar residues are colored yellow, nonpolar residues are colored white, acid residues are colored red, and basic residues are colored blue. F, differential electrostatic repulsion forces between the bound hBD2 and S4 segment affecting the activation of wild type and mutant Kv1.3 channels. These differential electrostatic repulsion forces resulted in different ΔV₅₀ values. The S1 and S2 helixes are colored lime green, the S3 and S4 helixes are colored mauve, the S5 and S6 helixes are colored yellow, the S1-S2 linker was colored red, and other linkers are colored black. hBD2 was represented by its molecular surface: the basic residues are shown in blue, the polar residues are shown in green, and the nonpolar residues are shown in white. Basic residues on the S4 helix were indicated with ⊕.

FIGURE 5.

Novel interaction between the Kv1.3 channel and hBD2. A, representative structures of the Kv channel modulators. hBD2 (Protein Data Bank code 1FD4) is from human endogenous tissue, charybdotoxin (ChTX, Protein Data Bank code 2CRD) is from scorpion venom, and hanatoxin (HaTX, Protein Data Bank code 1D1H) is from spider venom. B, model of the transmembrane topology of the Kv channel highlighting residues that are crucial for Kv channel modulators. The pore blocker charybdotoxin interacting with the channel pore region (6), and voltage-modulator toxins, such as hanatoxin, target S3-S4 linker to modify the gating properties (5). hBD2 not only targets the channel pore region but also targets the channel S1-S2 linker for influencing channel activation and inhibiting channel currents. Vivid green, dark green, and pink represent hBD2, charybdotoxin, and hanatoxin, respectively.