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Männikkö R
,
Shenkarev ZO
,
Thor MG
,
Berkut AA
,
Myshkin MY
,
Paramonov AS
,
Kulbatskii DS
,
Kuzmin DA
,
Sampedro Castañeda M
,
King L
,
Wilson ER
,
Lyukmanova EN
,
Kirpichnikov MP
,
Schorge S
,
Bosmans F
,
Hanna MG
,
Kullmann DM
,
Vassilevski AA
.
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Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.
Fig. 1. NaV channel organization and NaV1.4 VSD-I sequence comparison with other voltage-gated channels. (A) Transmembrane topology of NaV channels. The S1–S4 helices are in blue, and the S5–S6 helices are in gray. Gating charges are marked by “+” signs, and those neutralized in HypoPP are marked by red diamonds. Colored frames indicate VSDs specifically targeted by gating modifier toxins, VSD-I studied by us is shown by magenta. (B) Spatial organization of NaV channels with one pore domain and four VSDs. (C) Alignment of NaV1.4 VSD-I with VSDs of other NaV and KV channels. Conserved aromatic/hydrophobic, charged, and polar residues are color-coded. Transmembrane segments are highlighted by gray background. The gating charge transfer center is marked by green asterisks. Conserved charged residues in the S4 helix are numbered. Mutation of R222 (red diamond) is associated with HypoPP. Residues of KV1.2/2.1 and NaV1.7-DII responsible for the interaction with hanatoxin (34) and huwentoxin-IV (35), respectively, are boxed. D, domain.
Fig. 2. Hm-3 inhibits IGPs in p.R222W and p.R222G. (A) Representative current traces of wild-type or p.R222W channels in Na+ or Na+/Gn+ solution. (B) IGP of wild-type (black, n = 20) and p.R222W (red, n = 21) channels in Na+ solution. (C) IGP for p.R222W (red, n = 21) and p.R222G channels (blue, n = 42) in Na+ (solid symbols) or Na+/Gn+ (open symbols) solution. (D) Current–voltage relationship of p.R222G IGP in the absence (blue) or presence (black) of 10 μM Hm-3 (n = 10). (Insets) Representative current traces are shown. Data were normalized to current amplitude in response to a step to −140 mV in the absence of Hm-3. [Scale bars: 50 ms (x), 1 μA (y).] (E) Dose–response curve of p.R222G IGP inhibition by Hm-3 at −80 mV (n = 3–10). I is the current measured in the presence of the Hm-3 concentration indicated in x axis. I(Control) is the current measured in its absence. (F) Current–voltage relationship of p.R222W IGP in the absence (red) or presence (black) of 10 μM Hm-3 (n = 4). Data were normalized as in D. Error bars show SEM, and dashed lines indicate the zero current level. Voltage protocols are described in SI Materials and Methods.
Fig. 3. VSD-I–specific action of Hm-3. (A) IGP in the absence (colored symbols) and presence (black symbols) of 10 μM Hm-3 for S4 mutant channels (p.R672G: n = 4, p.R1132Q: n = 4, p.R219G: n = 6, and p.R225G: n = 9). Leak-subtracted data are shown for all but p.R225G, for which raw data are presented. Data were normalized to current amplitude in response to a step to −140 mV in the absence of Hm-3. (B–D) INa inhibition by Hm-3; the number of experiments is given in Fig. S2. (B) Current–voltage relationship for wild-type channels in the absence (●) and presence of 1 μM (△) or 10 μM (○) Hm-3. (C) Representative current traces in response to a voltage step to −20 mV in the absence (solid) and presence (dashed) of Hm-3 for wild-type and p.R222G channels. [Scale bars: 2 ms (x), 0.5 μA (y).] (D) Percentage of current inhibited by 1 μM (Left) and 10 μM (Right) Hm-3 at −20 mV for wild-type (black) and mutant channels (*P < 0.01). Error bars show SEM, and dashed lines indicate the zero current level.
Fig. 4. Hm-3/VSD-I interaction interface. (A and B) Superposition of 1H,15N-TROSY spectra of VSD-I and Hm-3 at different VSD-I/Hm-3 molar ratios. (C) Interfaces of Hm-3 interaction with the micelle (blue) and VSD-I (magenta) are mapped on the Hm-3 structure. Disulfide bonds are colored in yellow. Gray mesh shows the approximate micelle surface with a radius of ∼24 Å. (D) Interface of VSD-I interaction with Hm-3 is mapped on a homology model of VSD-I. The side chains forming the interaction interface are annotated. The conserved Arg/Lys residues of the S4 helix are also shown. Hydrophobic aliphatic and aromatic residues, polar uncharged, positively charged, and negatively charged residues are colored in green, magenta, blue, and red, respectively. (E) Conductance–voltage relationships for the chimeric KV2.1 constructs containing the S3–S4 helix–loop–helix motif of one of the four NaV1.4 VSDs before (black) and following (colored) addition of 1.5 μM Hm-3 (n = 3 each, error bars show SEM). D, domain; G is the conductance measured at the voltage indicated in x axis, Gmax is the normalized maximal conductance measured in control condition.
Fig. 5. Mode of Hm-3 action on IGP. (A) Model of the Hm-3/VSD-I complex. The backbones of fragments that participate in the formation of interaction surfaces are colored in red (VSD-I) and magenta (Hm-3). The charged and hydrophobic side chains at the interface are annotated. Salt bridges are shown by dotted lines. The micelle surface is shown by a dashed line. (B) Position of Hm-3 relative to the full-length α-subunit of NaV channel. (C) VSD-I of NaV1.4 channel modeled in the up and down states. The Hm-3 binding regions are colored red. Negatively and positively charged residues of the S3–S4 loop and S4 are colored red and blue, respectively. D, domain.
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