CgNa, a type I toxin from the giant Caribbean sea anemone Condylactis gigantea shows structural similarities to both type I and II toxins, as well as distinctive structural and functional properties(1)

Abstract

CgNa (Condylactis gigantea neurotoxin) is a 47-amino-acid- residue toxin from the giant Caribbean sea anemone Condylactis gigantea. The structure of CgNa, which was solved by 1H-NMR spectroscopy, is somewhat atypical and displays significant homology with both type I and II anemone toxins. CgNa also displays a considerable number of exceptions to the canonical structural elements that are thought to be essential for the activity of this group of toxins. Furthermore, unique residues in CgNa define a characteristic structure with strong negatively charged surface patches. These patches disrupt a surface-exposed cluster of hydrophobic residues present in all anemone-derived toxins described to date. A thorough characterization by patch-clamp analysis using rat DRG (dorsal root ganglion) neurons indicated that CgNa preferentially binds to TTX-S (tetrodotoxin-sensitive) voltage-gated sodium channels in the resting state. This association increased the inactivation time constant and the rate of recovery from inactivation, inducing a significant shift in the steady state of inactivation curve to the left. The specific structural features of CgNa may explain its weaker inhibitory capacity when compared with the other type I and II anemone toxins.

Figure 1. Stereo view of the 20 representative structures of CgNa

The structures are superimposed over the backbone atoms N, C^α and C′ of residues 1–47.

Figure 2. Structure-based sequence alignment of the CgNa toxin with type I (above CgNa) and type II (below CgNa) sea-anemone polypeptides of known structure

Following the notation from Norton [4], identical residues in each class are boxed. Those residues that are conservatively substituted are shaded in grey. *, Residues that are identical in all type I and type II sequences shown, †, Residues which are conservatively substituted. The residue numbering scheme is based on the CgNa sequence. ApA and ApB have a Pro-Ser insertion following Gly²⁵, causing the numbering of the residues after this position to be shifted by two units in relation to CgNa.

Figure 3. Ribbon structure representation and space-filling models of CgNa, ApB and Sh I toxins

(A) Superimposed ribbon representation of CgNa and ApB (left) and CgNa and Sh I (right) structures. CgNa is coloured green and dark grey. For CgNa and ApB, the structure that is closest to the average is shown, while for Sh I the closest to the average refined minimized structures of Wilcox et al. [56] is shown. (B) Solvent-accessibility surface representations of ApB (left), CgNa (middle) and Sh I (right), which represents an approx. 90° counterclockwise rotation around the vertical axis of the structures shown in (A). Electrostatic potential at the surface is represented by graded colours (blue, highly positive; red, highly negative). (C) Solvent-accessibility of the surface of ApB (left), CgNa (middle) and Sh I (right). Hydrophobic residues are coloured yellow, and positively and negatively charged amino acids are blue and red respectively. The views are rotated approx. 180° counterclockwise around the vertical axis compared with those in (A).

Figure 4. Current- and voltage-clamp experiments on TTX-S voltage-gated sodium channels in the presence of CgNa

(A) CgNa increases the length of the action potential. Action potentials of a DRG neuron recorded under control conditions, in the presence of CgNa (10 μM) and after washout (5 min). In this particular cell, a small depolarization of approx. 2 mV was observed. The dotted line in all panels indicates the zero voltage level. Inset: data for the entire experimental group in which current-clamp experiments were carried out. Exposure to the toxin increased the duration of action potentials measured at 50% of its amplitude by 463% (n=3). (B) CgNa increased the inactivation time course of TTX-S I_Na. Inset: representative experiment showing the effect of CgNa (10 μM) 1 min after commencing perfusion. Note the slowing of the inactivation process. In both cases, the current level returned to zero after the end of the depolarization pulse. The main plot shows the dose–response curve for CgNa (n=41) on τ_h. Data were fitted (continuous line) by a dose–response function with an IC₅₀ of 1.34±0.4 μM (n=41) and a Hill slope coefficient of 0.6±0.2 (n=41). (C) Effect of CgNa on the current-density–voltage curve. Top: I_Na produced at different potentials in the presence and absence of CgNa (10 μM, for clarity only the first 5 ms of each record are shown). Currents were produced by voltage pulses to the potentials indicated from a holding potential of −100 mV. Note that CgNa affects the currents at all the voltages tested. Bottom: current-density–voltage relationships (n=6). Perfusion with CgNa (10 μM, closed circles) did not produce significant changes either on the voltage at which the maximum current density was reached or on the reversal potential. (D) Effect of CgNa on steady-state inactivation. Top: representative experiment from which the curves were obtained. Bottom: steady-state inactivation of I_Na (n=6). The steady-state inactivation parameter (h_∞) was determined using the two-pulse protocol shown in the inset. Data obtained at the test pulse were plotted as a function of the pre-pulse potential and fitted to a Boltzmann function (continuous lines). CgNa (10 μM, closed circles) caused a significant 8 mV hyperpolarizing shift in the V_½,inact. The slope factor was also significantly changed at 3.5 mV.

Figure 5. CgNa enhanced the time course of recovery from inactivation

(A) Recovery curves obtained from the protocol in the inset (V_h=−100 mV). Curves were fitted (continuous lines) to a second order exponential function with a fast (τ_rec,fast) and a slow (τ_rec,_slow) time constant. CgNa (10 μM, closed circles) did not produce significant changes under these parameters. (B) Using V_h=−80 mV, the τ_rec,slow decreased significantly from 49.6±8.2 ms in control to 20.8±3.5 ms in the presence of CgNa.

Figure 6. CgNa acts on TTX-S voltage-gated sodium channels in the closed state

(A) Post-resting protocol employed to obtain the records. The toxin was applied at the beginning of the rest period (1 min) represented by the break. (B) Temporal course of the inactivation time constant τ_h. These data were obtained from a single cell, and it can be observed that the maximum effect of CgNa was already evident from the first pulse after the rest period. The bar indicates perfusion with 10 μM CgNa.

Figure 7. CgNa did not alter TTX-R sodium, potassium or calcium currents

In all panels, two superimposed traces and two current-density–voltage curves are presented, one under control conditions (open circles) and the other approx. 2 min after toxin perfusion (closed circles). (A) TTX-R sodium currents were isolated in the presence of 300 nM TTX. (B) Current-density–voltage curves for the potassium channels were obtained measuring the maximum current (circles) or the current at the end of the trace (squares) and normalizing these data to membrane capacity. (C) Current-density–voltage curves as obtained for calcium channels.