Brevetoxin versus Brevenal Modulation of Human Nav1 Channels

Abstract

Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-clamp. While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is >1000-fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels' interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.

Keywords: Nav; PbTx-2; PbTx-3; brevenal; brevetoxins; ladder-frame polyethers; voltage-gated sodium channels.

Figure 1

Structure of brevetoxins PbTx-3 (gold), PbTx-2 (magenta), and brevenal (blue). The six-membered ring lactone (Head) and side chain (Tail) of the brevetoxins are shown within gray boxes.

Figure 2

PbTx-3 and Brevenal differentially modulate Na⁺ currents mediated by human neuronal and muscle Nav channels. Representative whole-cell current traces mediated by Nav1.2 (A), Nav1.7 (B), Nav1.4 (C), and Nav1.5 (D) channels recorded by automated patch-clamp (APC) exposed to increasing concentrations of PbTx-3 (gold, 10⁻¹²–10⁻⁶ M) and brevenal (blue, 10⁻⁹–10⁻⁵ M); control is shown in black. Insets display scaled traces to highlight current kinetics. Test pulse: 25 ms, −20 mV, Vh −120 mV, 0.1 Hz. Peak and sustained currents are indicated by the gray shading.

Figure 3

Differential potency of PbTx-3 and brevenal in the modulation of human Nav channel-mediated currents. (A) Nav1.2, (B) Nav1.7, (C) Nav1.4 and (D) Nav1.5. ▼ PbTx-3 I_peak; ■ PbTx-3 I_late; ▽ Brevenal I_peak; ☐ Brevenal I_late. Data represent mean ± SEM, n = 5 for all determinations.

Figure 4

PbTx-3 and brevenal differentially modulate the kinetics of neuronal Nav-mediated currents. (A,C) Representative whole-cell current traces (top) and I-V plots (bottom) in response to a standard stimulation protocol (25 ms, −100 to +60 mV, Vh −120 mV, 0.1 Hz) from recombinant Nav1.2 and Nav1.7, respectively, recorded in different experimental conditions: i. control intracellular and extracellular absence of toxin (Ctro: black); ii. control intracellular and extracellular PbTx-3o (gold, ▼ 1 nM, ▽ 1 μM), or Brevenalo (blue, 10 μM); iii. brevenal in intracellular without toxin in extracellular (Brevenali: gray); iv. brevenal in intracellular and PbTx-3 in extracellular (PbTx-3o/Brevenali: magenta). Nav1.4: Brevenali 0.1 μM, PbTx-3o 1 nM; Nav1.5: Brevenali 1 μM, PbTx-3o 1 μM. Scale bars: 1 nA, 5 ms. (B,D) Scatter plots of the relative maximal macroscopic conductance (Gmax^Tox/Gmax^Ctr, top), and shift in the voltage dependence of activation (V0.5^Tox − V0.5^Ctr in mV, bottom) of peak currents mediated by Nav1.2 (B) and Nav1.7 (D) in the presence of PbTx-3o (■), Brevenalo (⬤), and PbTx-3o/Brevenali (▲). One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ‡ p < 0.0001.

Figure 5

PbTx-3 and brevenal differentially modulate the kinetics of muscle Nav-mediated currents. (A,C) Representative whole-cell current traces (top) and I-V plots (bottom) in response to a standard stimulation protocol (25 ms, −100 to +60 mV, Vh −120 mV, 0.1 Hz) from recombinant Nav1.4 and Nav1.5, respectively, recorded in different experimental conditions: i. control intracellular and extracellular absence of toxin (Ctro: black); ii. control intracellular and extracellular PbTx-3o (gold, ▼ 1 nM, ▽ 1 μM) or Brevenalo (blue, 10 μM); iii. Brevenal in intracellular without toxin in extracellular (Brevenali: gray); iv. brevenal in intracellular and PbTx-3 in extracellular (PbTx-3o/Brevenali: magenta). Nav1.4: Brevenali 0.1 μM, PbTx-3o 1 nM; Nav1.5: Brevenali 1 μM, PbTx-3o 1 μM. Scale bars: 1 nA, 5 ms. (B,D) Scatter plots of the relative maximal macroscopic conductance (Gmax^Tox/Gmax^Ctr, top), and shift in the voltage dependence of activation (V0.5^Tox − V0.5^Ctr in mV, bottom) of peak currents mediated by Nav1.4 (B) and Nav1.5 (D) in the presence of PbTx-3o (■), Brevenalo (⬤), and PbTx-3o/Brevenali (▲). One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ‡ p < 0.0001.

Figure 6

Membrane (top) and extracellular views of brevetoxin PbTx-2 docked into the cryo-EM structure of the Nav1.2 channel. The channel repeats I, II, III, and IV are pink, yellow, green, and gray, respectively. Brevetoxin is depicted with cyan carbons and red oxygens in stick form. Brevetoxin-sensing residues detected through ASM [12] are colored according to their respective backbones. Residues that are in direct contact with PbTx-2 are shown as sticks or spheres (Gly). Thick sticks represent residues whose alanine substitutions cause a significant impact on brevetoxin with small confidence intervals, whereas thin sticks represent residues whose replacement by alanine causes Kd changes with large confidence intervals. Residues that have a significant impact on brevetoxin binding with small confidence intervals but are not in direct contact with the ligand are shown as lines.