Phyla- and Subtype-Selectivity of CgNa, a Na Channel Toxin from the Venom of the Giant Caribbean Sea Anemone Condylactis Gigantea

Abstract

Because of their prominent role in electro-excitability, voltage-gated sodium (Na(V)) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related Na(V) subtypes, making them powerful tools to study Na(V) channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive Na(V) currents in rat dorsal root ganglion neurons. To illuminate the underlying Na(V) subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (Na(V)1.2-Na(V)1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNa(V)1.3/β(1), mNa(V)1.6/β(1) and, to a lesser extent, hNa(V)1.5/β(1), while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNa(V)1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect Na(V) channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific Na(V) channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of Na(V) channel inactivation.

Keywords: inactivation; sea anemone; selectivity; sodium channel; subtype; toxin.

Figure 1

Effects of CgNa on cloned mammalian Na_V channel subtypes Na_V1. 2–Na_V1.8/β₁ expressed in Xenopus oocytes. The tested mammalian isoforms originate from rat (r), human (h), or mouse (m). Left-hand panels show representative whole-cell current traces in control (black traces) and in presence of 10 μM CgNa (gray traces). Middle panels show normalized current–voltage relationships (n = 3–7) in control (●) and in presence of 10 μM CgNa (○). Right-hand panels show steady-state activation and inactivation curves (n = 3–7) in control (● and ■, respectively) and in presence of 10 μM CgNa (○ and □, respectively), fit with the Boltzmann equation.

Figure 2

Effects of CgNa on the cloned insect Na_V channel DmNa_V1/tipE expressed in Xenopus oocytes. (A,B) Left-hand panels show representative whole-cell current traces in control (black traces) and in presence CgNa (gray traces) at a concentration of 100 nM (A) and 10 μM (B); middle panels show normalized current–voltage relationships (n = 3–6) in control (●) and in presence of CgNa (○) at a concentration of 100 nM (A) and 10 μM (B); right-hand panels display steady-state activation and inactivation curves (n = 3–6) in control (● and ■, respectively) and in presence of CgNa (○ and □, respectively) at a concentration of 100 nM (A) and 10 μM (B). (C) Time course of the increase (n = 6) in peak I_Na (Δ, 100 nM; ▲, 10 μM) and sustained I_Na measured after 100 ms (○, 100 nM; ●, 10 μM). (D) Dose–response curves displaying the concentration dependence of the CgNa-induced increase in I_Na peak (●) and sustained I_Na measured after 100 ms (▲). (E) Recovery from inactivation (n = 7) in control (●) and in the presence of 100 nM CgNa (○).

Figure 3

Dose–response relationships of the effects of CgNa on the insect and mammalian Na_V channels. Dose–response curves are constructed using the following three parameters to quantify the effects induced by CgNa: increase in peak I_Na (A), increase in I_Na 5 ms (B), and increase in I_Na 100 ms (C). Left-hand graphs show phyla-selectivity between insect and mammalian Na_V channels, while right-hand graphs zoom in to show the subtype-selectivity among the mammalian Na_V subtypes. (D,E) Bar diagram illustrating the differences in potency and efficacy of CgNa on the variety of assayed insect and mammalian Na_V channels.

Figure 4

Sequence alignment of IVS3–S4 from insect and mammalian Na_V subtypes. Amino acid alignment of the transmembrane segments S3 and S4 from the homologous repeat DIV connected by the extracellular loop. The two sequences on top are from insect channels (MdNa_V1 and DmNa_V1; Md, house fly; Dm, fruit fly), the other sequences are from mammalian channel subtypes (Na_V1.1–Na_V1.9; r, rat; m, mouse; h, human). The Na_V channel subtypes affected by CgNa are shown on a gray background, residues that differ to those in the sequence of Na_V1.1–Na_V1.3 are colored red. Amino acid residues discussed in the text are printed in bold. Positions of the outermost N-terminal residues in the aligned sequence of each subtype are: MdNa_V1, 1668 (Uniprot accession number Q94615); DmNa_V1, 1680 (P35500); rNa_V1.1, 1602 (P04774); rNa_V1.2, 1592 (P04775); rNa_V1.3, 1538 (P08104); rNa_V1.4, 1407 (P15390); hNa_V1.5, 1589 (Q14524); mNa_V1.6, 1581 (Q9WTU3); rNa_V1.7, 1574 (O08562); rNa_V1.8, 1538 (Q62968); rNa_V1.9, 1407 (O88457).