Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.
???displayArticle.pubmedLink???
39042091
???displayArticle.pmcLink???PMC11270453 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure S1. Electrophysiology protocols used and NaV1.6 inactivation. (A) Shown are illustrations of the voltage-step protocols used in experimental work. (B) 100 nM GrTX-SIA does not influence the inactivation rate of NaV1.6. Representative current traces at −30 and −10 mV are shown before (black) and after (red) a 4-min incubation period with 100 nM GrTX-SIA. A single exponential fit of the inactivation phase yielded a time constant (τ) that is plotted against its voltage step in the right panel. Circles represent mean ± standard error of the mean (SEM) of n = 6 oocytes.
Figure 1. GrTX-SIA inhibits multiple NaV
channel subtypes. (A–C) Representative current traces (A), normalized conductance (G/Gmax)–voltage and channel availability (I/Imax) relationships (B), and recovery from inactivation (% rec) are shown before (black) and after (red) a 4-min incubation period with 100 nM GrTX-SIA (C). Data were normalized by comparing the saturated toxin effect to the corresponding control recordings obtained before toxin addition to the same oocyte. The holding potential was −90 mV with 5 s between depolarizing pulses, except for NaV1.8, which was measured with 10 s between pulses (protocol shown in Fig. S1). Traces shown were recorded at −15 mV for NaV1.1, NaV1.3, NaV1.4, and NaV1.7, −20 mV for NaV1.2 and NaV1.6, −30 mV for NaV1.5, and +10 mV for NaV1.8. X-axis represents 10 ms for all traces, Y-axis value is indicated. Fit values are reported in Table S1. Circles represent mean ± SEM of n = 6 oocytes for NaV1.1, NaV1.5, NaV1.6, NaV1.7, and NaV1.8, n = 5 oocytes for NaV1.2 and NaV1.4, and n = 4 oocytes for NaV1.3.
Figure 2. GrTX-SIA inhibits NaV1.6. (A–C) Representative current trace recorded at −20 mV (A), normalized conductance (G/Gmax)-voltage and channel availability (I/Imax) relationships (B), and recovery from inactivation (% rec; C) are shown before (black) and after (red) a 4-min incubation period with 1 μM GrTX-SIA. The holding potential was −90 mV with 5 s between depolarizing pulses (see Fig. S1). (D) Dose-response relationship, fitted with the Hill equation, of GrTX-SIA (concentration in nM) versus percentage of current remaining when measured at −10 mV. Circles represent mean ± SEM of n = 6 oocytes for 1 nM, 10 nM, 100 nM, and 1 µM, and n = 5 oocytes for 3 µM.
Figure S2. GrTX-SIA and NaV1.6 slow inactivation. 1 µM GrTX-SIA affects the voltage-dependence of slow inactivation (left) but not entry into slow inactivation (right). Control is shown in black and toxin condition in red. Circles represent the mean ± SEM of n = 8 oocytes for voltage-dependence of slow inactivation and n = 5 oocytes for entry into slow inactivation; *P < 0.05 compared to without toxin.
Figure 3. GrTX-SIA inhibition of NaV1.6 currents. GrTX-SIA application (1 µM) leads to a quick initial inhibition (red traces) followed by a somewhat slower and smaller inhibitory step (grey/blue). This effect was variable between measurements and only served as a rationale to investigate the potential presence of multiple toxin binding sites within NaV1.6. (A and B) Panel A shows a representative trace, whereas panel B displays a typical NaV1.6 current that is inhibited (Y-axis: percentage of current remaining assessed with 5 s pulses to −25 mV) in two phases during a 4-min application of 1 µM GrTX-SIA. The second phase is not related to channel current rundown. Current amplitude partially recovered during a 5-min wash-off period (black circles in B). (C) The normalized G–V (G/Gmax) and channel availability (I/Imax) relationships are shown for the starting condition without toxin (black), after the first inhibitory phase (red) and after the remaining slow step (grey/blue). Currents were recorded by applying a depolarizing voltage-step protocol from −100 to +20 mV from a holding potential of −90 mV with 5 s between depolarizing pulses. Circles represent the mean ± SEM with n = 6 oocytes.
Figure 4. GrTX-SIA interacts with two KV2.1/NaV1.6 VSD chimeras. (A) Example potassium ion currents (top row) elicited by depolarizations to the maximum voltage of the voltage-activation curve (second row) for the four KV2.1/NaV1.6 VSD chimeras (VSDI–VSDIV) in the absence (black) and presence of 1 µM GrTX-SIA after a 4-min incubation period. The holding voltage was −90 mV and the tail voltage was −60 mV (−80 mV for DIII). The second row of panel A shows the normalized current–voltage relationships for the corresponding KV2.1/NaV1.6 VSD chimeras before and after the addition of 1 µM GrTX-SIA, normalized to the maximal control current. *P < 0.05 compared with recordings before toxin addition. (B) Representative example of partial KV2.1/NaV1.6 VSDII inhibition (left) by 1 µM GrTX-SIA followed by wash-off with ND-100 (Y-axis: percentage of current remaining assessed with 5 s pulses to +50 mV). Current amplitude completely recovered during a 4-min wash-off period (black circles). Right panel shows virtually complete KV2.1/NaV1.6 VSDIV inhibition by 1 µM GrTX-SIA followed by incomplete wash-off with ND-100 after 10 min. Data shown are mean ± SEM of n = 7 oocytes for KV2.1/NaV1.6 VSDI, and n = 5 oocytes for KV2.1/NaV1.6 VSDII–VSDIV.
Figure S3. GrTX-SIA interacts with VSDII and VSDIV in NaV1.6. Shown are representative current traces of KV2.1/NaV1.6 VSDII (top) and KV2.1/NaV1.6 VSDIV (bottom) before (black; control) and during 1 µM GrTX-SIA application (red) for 4 min. Grey traces indicate recovery of ionic currents upon toxin washout with 50K solution. These data underscore the notion that GrTX-SIA inhibited the KV2.1/NaV1.6 VSDII chimera more slowly compared to KV2.1/NaV1.6 VSDIV. Moreover, current recovery was faster for the KV2.1/NaV1.6 VSDII chimera compared with KV2.1/NaV1.6 VSDIV.
Figure 5. AlphaFold predictions for GrTX-SIA binding to human NaV1.6. (A) Cryo-EM structure of human NaV1.6 (PDB accession no. 8FHD) with proposed docking sites for GrTX-SIA (transparent red) on VSDII and VSDIV. Shown is a view from within the plane of the plasma membrane. The NaVβ1 subunit is shown in green. VSDs of the channel are shown in blue and the remainder in grey. GrTX-SIA binding positions are obtained by superposition of AlphaFold models for GrTX-SIA bound to VSDII and VSDIV individually. (B) Same as in panel A, but shows a top view from the extracellular space facing the plasma membrane. (C) Top AlphaFold model for GrTX-SIA (red) bound to VSDIV (blue). Select residues are labeled. (D) AlphaFold model for GrTX-SIA (red) bound to VSDII (blue). This model corresponds to #6 in the AlphaFold model ranking, but is the highest ranked model whereby GrTX-SIA is bound to the extracellular part of VSDII. Select residues are labeled.
Figure 6. Mutations in NaV1.6 VSDII and VSDIV influence GrTX-SIA binding. The effect of 1 µM GrTX-SIA on NaV1.6 VSDIVmut is drastically reduced compared to the wild-type channel. The top row shows a representative trace before (black) and after (red) toxin application for 4 min, the normalized G-V (G/Gmax) and channel availability (I/Imax) relationships under these conditions, and a representative example of partial NaV1.6 VSDIVmut inhibition by 1 µM GrTX-SIA followed by complete wash-off with ND-100 after 4 min. The bottom row displays the virtual lack of effect of 1 µM GrTX-SIA on the NaV1.6 VSDIImut–VSDIVmut construct. In both panels, circles represent the mean ± SEM of n = 8 oocytes for NaV1.6 VSDIVmut and 5 oocytes for NaV1.6 VSDIImut–VSDIVmut, with n = 3 for toxin wash-in/wash-off experiments.
