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

Background: The functional role of the Kv channel S1-S2 linker remains unclear. Results: The S1-S2 linker-hBD2 interaction modifies Kv1.3 channel activation through electrostatic repulsion between positively charged hBD2 and the channel S4 segment. Conclusion: The Kv1.3 channel S1-S2 linker is a novel peptide-binding site. Significance: These findings could define the function of S1-S2 linkers in Kv channel gating modification among different Kv channels. 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.

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 ␤-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 ϳ31.2 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.
Voltage-gated K ϩ (Kv) 4 channels are molecular sensors of membrane potential (1) and play critical roles in cellular signal-ing in both neuronal and non-neuronal cells. Kv channels are tetramers, and each subunit comprises six membrane-spanning helical segments, named S1-S6, as well as three extracellular domains: the S1-S2 linker, the S3-S4 linker, and the pore loop (2). During the past years, animal toxins, which are selective and potent molecular ligands, have been proven to be invaluable in unraveling Kv channel structure and functions. In general, animal toxins target Kv channels through two distinct extracellular domains: channel pore-blocking toxins recognize the extracellular pore region, and channel gating-modifier toxins interact with the extracellular S3-S4 linker (3)(4)(5)(6). However, whether the extracellular S1-S2 linker is involved in the recognition of peptide ligands remains unclear.
Recently, we identified human ␤-defensin 2 (hBD2) as an animal toxin-like endogenous Kv1.3 channel inhibitor that can interact with extracellular pore region (6,7). Surprisingly, we found that hBD2, unlike the classic channel pore-blocking toxins that generally do not affect Kv channel kinetics (8,9), works simultaneously as a gating modifier and shifts the conductancevoltage relationship (G-V) curve of the Kv1.3 channel to a more depolarized voltage by nearly 10 mV. Additionally, hBD2 directly interacts with the Kv1.3 channel S1-S2 linker but not the S3-S4 linker. These findings indicate that the Kv1.3 channel S1-S2 linker is critically involved in hBD2 modification of Kv1.3 channel activation.
QuikChange Lightning multi site-directed mutagenesis kit (Stratagene) based on a wild type Kv1.2 plasmid. All plasmids were verified with DNA sequencing before protein expression.
Cell Cultures-HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified 5% CO 2 incubator at a temperature of 37°C. The cells were transfected using FuGENE transfection reagent (Roche Diagnostics) following the manufacturer's instructions, and the cells were used for electrophysiology 24 -48 h after transfection.
Electrophysiology Recording and Data Analyses-Electrophysiological experiments were performed at 22-25°C using a whole cell patch clamp recording mode with a HEKA EPC 10 amplifier and Patchmaster data acquisition software as described previously (8,10). The cells were bathed with mammalian Ringer's solution: 5 mM KCl, 140 mM NaCl, 10 mM HEPES, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM D-glucose (pH 7.4 with NaOH). 0.01% BSA was added to the Ringer's solution when the hBD2 was applied. The pipette solution contained: 140 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 1 mM Na 2 ATP, 5 mM HEPES (pH 7.2 with KOH). Channel currents were elicited with depolarizing voltage steps of 200 ms from a holding potential of Ϫ80 to ϩ50 mV to investigate hBD2 affinity. The kinetics of activation of the wild type and mutant channels were investigated by eliciting currents from a holding potential of Ϫ80 mV to test potentials in a range from Ϫ60 to ϩ40 mV (5-mV increments, 200 ms in duration). The currents were typically digitized at 20 kHz and filtered at 2.9 kHz (Bessel). 85% of the series resistances were electronically compensated. The data were analyzed with Patchmaster, Clampfit (Axon Instruments, Inc.), and SigmaPlot (SPSS Inc.). The IC 50 values were obtained by fitting a modified Hill equation to the data with the following formula: I hBD2 /I control ϭ 1/(1/([hBD2]/IC 50 ), where [hBD2] is the concentration of hBD2, and I hBD2 and I control are the peak currents in the absence and presence of hBD2 at five different concentrations. The conductance of the Kv1.3 channels was calculated from G ϭ I peak /(V Ϫ E k ), where I peak is the peak current, and E k is the reversal potential of the Kv1.3 channel. G-V curves were generated using the measured peak currents and fitted with a Boltzmann equation using the following formula: G/G max ϭ 1/(1 ϩ exp[(V Ϫ V 50 )/k]), where G is the conductance of the channels, G max is the maximal channel conductance, V is the membrane voltage, V 50 is the voltage of halfmaximal activation, and k is the slope factor. The activation kinetics were determined using the dominant time constant of activation approach, in which a single exponential was fitted to the late rising phase of the raw current data using Clampfit (Axon Instruments, Inc.) to calculate a fourth power function with the following formula: where I(t) represents the macroscopic and time-dependent current, A is the current predicted at steady state, is the time constant, and C is a constant. The data are presented as the means Ϯ S.D. of at least three experiments.
Co-immunoprecipitation Assay-Cells transfected with FLAG-tagged Kv1.3, Kv1.2 and the S1-S2 linker chimera channels were washed with ice-cold PBS and extracted using the Transmembrane protein extraction kit according to the manufacturer's instructions (Merck). Precleared protein was obtained by incubating the cell supernatants with a 40-l protein G-Sepharose bead slurry (GE Healthcare) for 1 h at 4°C to reduce nonspecific binding of proteins to the Sepharose beads. After centrifugation, adequate soluble hBD2 was added to the supernatant and incubated under rotation for 4 h at a temperature of 4°C. Subsequently, 50 l of the protein G-Sepharose beads and 5 l of an anti-FLAG antibody (Sigma) were added to the samples, and rotation was continued overnight. After the overnight incubation, the beads were washed three times with ice-cold modified radioimmune precipitation assay buffer, followed by resuspension of the Sepharose beads in 2ϫ SDS sample buffer. The samples were separated by SDS-PAGE and then transferred onto Immobilon-P membranes (Millipore) for Western blot analysis.

hBD2 Regulates Kv1.3 Channel Activation Kinetics-hBD2
recruits T cells and influences the cytokine secretion of T cells through interactions with different receptors (7, 11, 12). Our recent report showed that hBD2 inhibits Kv1.3 channel currents by interacting with the channel extracellular pore region (7). Surprisingly, the Kv1.3 currents were more significantly inhibited by hBD2 at Ϫ30 mV than at 0 mV ( Fig. 1, A and B), which suggests that in addition to serving as a channel pore blocker, hBD2 could also regulate Kv1.3 activation kinetics. In contrast to the voltageindependent inhibition of Kv1.3 channel currents by channel pore-blocking toxins (3,8,13), voltage-dependent inhibition is a hallmark of gating-modifier toxins for Kv channels, which suggests that hBD2 has other effects on Kv1.3 in addition to being a channel pore blocker. Consistent with a channel gating modifier, hBD2 shifted the midpoint of activation (V 50 ) of the Kv1.3 activation G-V 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 Ϯ 2.7% of the S5-S6 linker chimera currents blocked by 10 nM hBD2 (B), 9.2 Ϯ 1.9% of the S3-S4 linker chimera currents blocked by 1000 nM hBD2 (C), and 54.2 Ϯ 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 IC 50 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.
curves toward the positive direction by nearly 10.5 mV (Fig. 1C). Furthermore, hBD2 significantly increased the activation time constant () of the Kv1.3 channel at different voltages (Fig. 1D). These results suggested that hBD2 modifies human Kv1.3 channel activation rather than blocking the channel pore.
The Kv1.3 Channel S1-S2 Linker Is the Binding Site of hBD2-Next, we explored the structural requirements for hBD2 regulation of the Kv1.3 channel activation. Based on the insensitivity of the human Kv1.2 channel to hBD2 (7), we constructed chimeric channels using the human Kv1.2 and Kv1.3 channels ( Fig.  2A). Because the differences in the amino acid sequences are mainly located in three extracellular domains (the S1-S2 linker, the S3-S4 linker, and the S5-S6 linker) and hBD2 is likely to interact with amino acid residues on the extracellular regions, we made chimeric channels by swapping these three regions between the human Kv1.2 and Kv1.3 channels (Fig. 2A). 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 V 50 values before and after hBD2 interacts with different channels. The ⌬V 50 ϭ V 50 (ϩhBD2) Ϫ V 50 (ϪhBD2).
Consistent with our previous findings that the Kv1.3 pore region is critical to hBD2 inhibition (7), the S5-S6 linker chimera in which the S5-S6 linker of Kv1.2 channel was replaced with the equivalent region from the Kv1.3 channel exhibited marked inhibition by 10 nM hBD2 (Fig. 2B). However, the potency of this channel (IC 50 ϭ 8.8 Ϯ 3.9 nM) was ϳ400-fold less than the potency of the wild type Kv1.3 channel (Fig. 2, E  and F), which suggests that other extracellular domains of the Kv1.3 channel might also be required for hBD2 inhibition of Kv1.3.
Although the S3-S4 linker is a well known binding interface for animal gating-modifying toxins (5,14,15), replacement of the S3-S4 linker of Kv1.2 channel with an equivalent region from the Kv1.3 channel resulted in a chimeric channel that lacked sensitivity to inhibition by hBD2 (9.2 Ϯ 1.9% at 1000 nM) (Fig. 2C), which suggests that the S3-S4 linker was not essential to hBD2 inhibition of the Kv1.3 channel.
Interestingly, the S1-S2 linker chimeric channel, produced by substituting the S1-S2 linker in the Kv1.2 channel with the corresponding Kv1.3 channel domain, exhibited potent inhibition by 1 nM hBD2 (Fig. 2D). The potency of hBD2 inhibition (IC 50 ϭ 0.58 Ϯ 0.11 nM) was ϳ26-fold less potent than inhibition of the wild type Kv1.3 channel and ϳ15-fold more potent than the S5-S6 linker chimeric channel (Fig. 2, E and F). Taken together, these results suggest that the Kv1.3 channel S1-S2 linker is a novel interaction site for hBD2. To further confirm the interaction between hBD2 and the S1-S2 linker of the Kv1.3 channel, a co-immunoprecipitation (Co-IP) assay was performed. We immobilized different FLAG-tagged channels on protein G-Sepharose beads and detected whether the channels could retain hBD2 through Western blotting. Our results showed that the Kv1.3 channel could retain hBD2 and Kv1.2 channels retained much less hBD2 than the Kv1.3 channels, which was consistent with our previous studies (7). When the Kv1.3 channel S1-S2 linker was transferred to the Kv1.2 channel, the binding affinity of hBD2 and the S1-S2 linker channel chimera was higher than the binding affinity for the Kv1.2 channel (Fig. 2G). This Co-IP assay further confirmed the interaction between hBD2 and the S1-S2 linker of the Kv1.3 channel.
Kv1.3 Channel S1-S2 Linker-hBD2 Interaction Modifies Channel Activation-Next, we asked whether the Kv1.3 channel S1-S2 linker-hBD2 interaction could affect channel activation. As shown in Fig. 3 (A and B), Kv1.2 channel activation could not be modified by hBD2; however, activation of the S1-S2 linker chimeric channel, which was produced by substituting the S1-S2 linker in the Kv1.2 channel with the equivalent Kv1.3 channel domain, was markedly influenced by hBD2, and the shift in the G-V curves, ⌬V 50 , was ϳ10.5 mV, which is similar to the shift of G-V curves from wild type Kv1.3 (Figs. 1C and  3E). Generally, the S3-S4 linker-gating modifier toxin interactions shift the Kv channel G-V curves (15)(16)(17). However, no significant shift of the G-V curves was observed for the S3-S4 linker chimeric channel in the presence of hBD2 (Figs. 3, C and  E), which is likely due to a much weaker interaction between this chimeric channel and hBD2 (Fig. 2C). Additionally, the channel pore region-hBD2 interaction had less effect on the activation curve of the S5-S6 linker chimera channel (Fig. 3, D  and E). These data clearly support a unique role for the S1-S2 linker in the modification of Kv1.3 activation by hBD2.
Functional Residues in the Channel S1-S2 Linker Influence hBD2 Binding-We used alanine scanning mutagenesis to further identify the amino acid residues in the Kv1.3 S1-S2 linker that are required for the interaction between hBD2 and Kv1.3. Table 1 shows the V 50 values of channel activation for the wild type and 23 mutant Kv1.3 channels in which each individual amino acid was replaced with alanine in the absence and presence of hBD2. In comparison with the ⌬V 50 value of 10.5 mV for wild type Kv1.3 modified by hBD2, the ⌬V 50 values of the nine mutant channels were significantly decreased by ϳ70 -91%, which suggests that residues, including Asp-209, Glu-210, Lys-211, Tyr-213, Gly-235, Ser-238, Phe-238, Asp-241, and Pro-242, played an essential role in the hBD2 interaction with the S1-S2 linker of Kv1.3. The ⌬V 50 values of the Kv1.3-F207A and Kv1.3-S232A channels were ϳ4.4 mV, which suggests that Phe-207 and Ser-232 moderately affect hBD2 interaction with Kv1.3. In contrast, the other 12 residues, including Glu-206, Arg-208, Asp-212, Ser-218, Gln-219, Asp-220, Ser-221, Glu-223, Ser-230, Arg-233, Ser-237, and Ser-240, had less effect on hBD2 interaction with Kv1.3 because the ⌬V 50 values of their respective mutant channels were much closer or similar to the ⌬V 50 value of the wild type Kv1.3 (Table 1). To verify the differential effects of the S1-S2 linker residues on hBD2 binding, we performed Co-IP experiments with three representative mutants, D209A, F207A, and D220A, and found remarkable, moderate, and small reductions in ⌬V 50 values, respectively. In line with their ⌬V 50 values, the channel mutants D209A, F207A, and D220A retained more and more hBD2 (Fig. 4C). Together, these results indicated that the amino acid residues in the Kv1.3 channel S1-S2 linker acted differentially to interact with hBD2, which resulted in modification of the channel activation kinetics.
Kv1.3 Channel Activation Modification by Electrostatic Repulsion between S4 and hBD2-Our results showed that hBD2 can anchor in two terminals of the Kv1.3 channel S1-S2 linker for channel activation modulation (Table 1 and Fig. 4, A  and E). Similar to other classical Kv channels, Kv1.3 activation from resting state is accompanied by a voltage-dependent outward movement of the S4 segment, which is enriched with con-served basic amino acid residues such as arginine and lysine (Fig. 4A) (18). We hypothesize that hBD2 modification of Kv1.3 channel activation is caused by an electrostatic repulsion between S4 and basic hBD2 anchoring in the S1-S2 linker (Fig.  4F). To test this hypothesis, we constructed three mutant Kv1.3 channels by altering the positive electric charges in the S4 segment. The Kv1.3-A309R channel has an additional arginine residue in the S4 N-terminal; the Kv1.3-R312H/R315H channel had two arginine residues adjacent to S4 N-terminal replaced by histidine residues that partially maintained the positive electric charges; and the Kv1.3-R312S/R315S channel had two arginine residues replaced by polar serine residues. When the electrostatic repulsion force between hBD2 and S4 was increased in the Kv1.3-A309R channel, the G-V curve was shifted to a much more depolarized voltage in the presence of hBD2 (Fig. 4B). The corresponding ⌬V 50 value was ϳ31.2 mV, which is much larger than the ⌬V 50 value of wild type Kv1.3 (Fig. 1). However, whereas the electrostatic repulsion force between hBD2 and S4 was decreased in the Kv1.3-R312H/R315H and Kv1.3-R312S/ R315S channels, there was no obvious shift in the G-V curves (Fig. 4, B and F). The ⌬V 50 values were ϳ4.4 mV and 2.2 mV for the Kv1.3-R312H/R315H and Kv1.3-R312S/R315S channels, respectively. In comparison with a ⌬V 50 value of 10.5 mV for the wild type Kv1.3 channel modified by hBD2 (Fig. 1), these ⌬V 50 values of the Kv1.3 S4 segment mutants corresponded to changes in the positive electric charges in the S4 segment of the mutant channels. In agreement with the minor effect of the S3-S4 linker on hBD2 binding (Fig. 2, C and F), the Co-IP experiments showed that all three S4 segment mutants with either more and fewer positive electric charges did not obviously impair hBD2 binding (Fig. 4D). These results highlight a critical role of electrostatic repulsion between hBD2 and S4 in Kv1.3 activation (Fig. 4F).

Discussion
The Kv Channel S1-S2 Linker Is a Novel Binding Site for a Peptide Ligand-The extracellular domains of Kv channels are critical interaction sites for both endogenous and exogenous molecules. Among the three extracellular domains of Kv channels, the functions of the S3-S4 linker and the pore region have been well documented by animal toxin studies in past years (19) (Fig. 5, A and B). The channel S3-S4 linker was targeted by the spider toxin hanatoxin (5), and the channel pore region was bound by the scorpion toxin charybdotoxin (3,20). However, whether the channel S1-S2 linker can be recognized by a peptide ligand remains unknown. Our present work showed that the channel S1-S2 linker is a novel binding site of the hBD2 ligand.
We demonstrated that the S1-S2 linker of the Kv1.3 channel was critically involved in hBD2 interaction and modification of the Kv1.3 activation. By constructing chimeric channels in addition to site-directed mutagenesis and Co-IP experiments, we showed that the S1-S2 linker was responsible not only for hBD2 binding (Fig. 2) but also for hBD2-elicited regulation of the Kv channel activation kinetics (Fig. 3). Furthermore, we identified 9 amino acid residues in the S1-S2 linker that are required for hBD2 modification of Kv1.3 activation (Table 1). Although both the S1-S2 linker and the S3-S4 linker are located  (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. 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 ⌬V 50 values was as follows: Asp-209 Ͼ Phe-207 Ͼ Asp-220. The binding affinity with hBD2 was: D209A Ͻ F207A Ͻ 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 V 50 , 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 ⌬V 50 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 Q.
in the vicinity of the Kv channel voltage sensor domain, the S1-S2 linker of the Kv1.3 channel is much longer than the S3-S4 linkers of the Kv2.1 or Kv4.3 channels that are targeted by gating-modifier toxins (Figs. 4F and 5B) (5,16,17,21), which likely results into different structural features of the channel receptor sites. Unlike residues in the C-terminal half of the S3 segment of the Kv channel S3-S4 linker, which generally affect toxin binding (21-23) (Fig. 5B), we found that essential amino acid residues required for hBD2 interaction with Kv1.3 S1-S2 linker are located at both terminals (Table 1 and Figs. 4 and 5). In fact, many residues in the middle of the S1-S2 linker had a minor effect on hBD2 interaction with Kv1.3 (Table 1 and Fig. 4). This feature of the hBD2-Kv1.3 interaction interface suggests that the Kv1.3 channel S1-S2 linker likely forms an extended loop similar to the loop in Kv1.2, which was observed by normalmode-based x-ray crystallographic refinement (24) (Fig. 4). Unlike the known roles of each Kv channel extracellular domain individually targeted by one type of animal toxin (Fig. 5,  A and B), both the S1-S2 linker and the pore region of Kv1.3 channel are binding sites for hBD2 (Fig. 5B), and the corresponding features of the channel-interacting interfaces in hBD2 would be interesting to explore.
The Kv Channel S1-S2 Linker Plays a Role of a Bridge Pier in Channel Activation Modulation-So far, no evidence shows an interaction between the S1-S2 linker and the S3-S4 linker in Kv channels. The structural analysis would help to depict the functional role of channel S1-S2 in the modulation of channel activation. The crystal structure of the Kv1.2 channel indicates that transmembrane S1, S2, S3, and S4 helices look like two rows of bridge piers with unknown signal communication (25) (Fig. 4F). When hBD2, which is a classical positively charged peptide ligand, binds both terminals of the channel S1-S2 linker, it is anchored in the S1-S2 bridge pier and shortens the spatial distance from the positively charged S4, which is strongly supported by the modification of Kv1.3 activation with increasing and decreasing positive charges in the channel S4 helix (Fig. 4). Therefore, the channel S1-S2 linker likely plays the novel role of a bridge pier in the modulation of Kv1.3 channel activation through electrostatic repulsion forces between the anchored hBD2 and the channel S4. Additionally, the preference of S1-S2 linkers as bridge piers between the Kv1.2 and Kv1.3 channels is an aspect of Kv channels that requires further investigation in the future.
In conclusion, we found a novel functional role for the Kv1.3 channel S1-S2 linker in channel gating as revealed by interactions between the linker and an endogenous gating-modifier, hBD2. Our work not only reveals a potent S1-S2 linker-hBD2 interaction and demonstrates its effect on Kv channel activation but also elucidates the distinct "electrostatic repulsion mechanism" underlying the modification of Kv1.3 channel activation. These findings might be important for defining the function of S1-S2 linkers in the modification of Kv channel gating among different Kv channels in future studies.