In Silico Simulations and Functional Cell Studies Evidence Similar Potency and Distinct Binding of Pacific and Caribbean Ciguatoxins

Abstract

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale. Graphical Abstract

Synergistic Effect of Brevetoxin BTX-3 and Ciguatoxin CTX3C in Human Voltage-Gated Nav1.6 Sodium Channels

“…The effects of increasing CTX3C concentrations, between 0.000001 and 10 nM, on the maximum peak inward sodium currents ( I Na ) and the activation voltage of human VGSC after single cell exposure were first evaluated and reported by our group . As previously reported, ,, CTX3C elicited a concentration-dependent decrease in the maximum peak amplitude of sodium currents being detected even at the lowest concentrations. In control conditions, the peak sodium current at −10 mV was −1156 ± 189 pA ( n = 13) decreasing in a concentration-dependent manner up to −692 ± 372 pA ( n = 3) after bath application of 1 nM CTX3C.…”

“…CTXs and BTXs bind to a common site of the sodium channel located at the cleft created by segment S5 of domain IV, segment S6 of domain I, and the P-loop (P1) that connects segments S5 and S6 of domain IV of the VGSC α-subunit , but with different affinities. Previous studies indicated that CTXs interact specifically and with higher affinity with sodium channels; , however, the differences in the chemical structures between the CTX analogues generate modifications in their binding and activity over VGSC. , BTXs are also considered to have a high binding affinity to sodium channels; , however, the differences between both groups of toxins are significant since studies of their binding affinity to rat brain sodium channels allowed to calculate an inhibitory constant (ki) of 0.041 nM for ciguatoxin-1B which was more than 50-fold higher than that of BTX-1 (2.24 nM) .…”

“…The functional consequence of the interaction of CTXs and BTXs with VGSC is a hyperpolarizing shift in the sodium channel activation curve ,, as well as a negative shift of their half inactivation voltage . CTXs are known to have a significant effect on the activation and inactivation state of VGSC at concentrations of 1 nM and higher. ,, As a consequence, the channels will remain permanently open at resting cell membrane potentials, resulting in depolarization of the membrane and spontaneous and/or repetitive action potential discharges in excitable cells. , Although both groups of toxins interact with the same site of VGSC, there are important differences between the observed cellular effect of each group since CTXs cause a remarkable decrease in the maximum peak inward sodium current amplitude ( I Na ), ,, while no significant effects were reported for BTXs. , It is important to note that the effects and the affinity of CTXs or BTXs for VGSC depend on the analogue and the channel subtype . Moreover, the binding of BTXs to sodium channels is highly state-dependent, thus, channel opening facilitates BTX binding, feeding back their binding and sodium influx …”

“…CTXs and BTXs bind to a common site of the sodium channel located at the cleft created by segment S5 of domain IV, segment S6 of domain I, and the P-loop (P1) that connects segments S5 and S6 of domain IV of the VGSC α-subunit 6 , 7 but with different affinities. Previous studies indicated that CTXs interact specifically and with higher affinity with sodium channels; 8 , 9 however, the differences in the chemical structures between the CTX analogues generate modifications in their binding and activity over VGSC.…”

“…The functional consequence of the interaction of CTXs and BTXs with VGSC is a hyperpolarizing shift in the sodium channel activation curve 5 , 7 , 15 as well as a negative shift of their half inactivation voltage. 16 CTXs are known to have a significant effect on the activation and inactivation state of VGSC at concentrations of 1 nM and higher.…”