Low concentrations of tetrodotoxin interact with tetrodotoxin-resistant voltage-gated sodium channels.
OBJECTIVE
Tetrodotoxin (TTX) is used to distinguish between two classes of voltage-gated sodium channel (VGSC)--TTX sensitive (TTXS) and TTX resistant (TTXR). The resistance of TTXR VGSCs is thought to result from a low binding affinity of TTX, although at high TTX concentrations channel block does occur. Here, we show that, at concentrations below those which produce block, TTX can bind to TTXR VGSCs.


METHODS
Whole-cell voltage clamp recordings were made from dissociated rat dorsal root ganglion neurones that expressed both TTXS and TTXR sodium currents. Voltage-gated calcium currents were blocked by 10 microM extracellular lanthanum chloride. TTXS, but not TTXR, current was suppressed by using a holding potential of -50 mV, and the effect of TTX on the isolated TTXR current was explored.


RESULTS
Extracellular application of 0.5 microM TTX produced a 40% increase in TTXR current amplitude, a negative shift in the voltage-dependence of current activation (approximately -8 mV) and inactivation (approximately -10 mV) and increased rates of current activation and inactivation. The effect of TTX on current amplitude was dose-dependent (EC50 = 364 nM). Removal of lanthanum prevented the effect of TTX on TTXR current amplitude, whereas reducing extracellular calcium did not.


CONCLUSIONS
The findings are consistent with an interpretation that TTX relieves a tonic block of the TTXR VGSC by lanthanum. We conclude that TTX binds to the TTXR VGSC at low concentrations, without blocking it. This appears to be the first demonstration of a clear distinction between binding affinity and blocking potency of a channel-blocking agent.
King's College London, Department of Clinical Neurosciences, London, UK. c.farmer@ion.ucl.ac.uk
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005727:Ganglia, Spinal / Q000187:drug effects / Q000378:metabolism; D007811:Lanthanum / Q000378:metabolism; D008564:Membrane Potentials; D062559:NAV1.8 Voltage-Gated Sodium Channel; D062558:NAV1.9 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000187:drug effects; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D026941:Sodium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D013779:Tetrodotoxin / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors
D062559:NAV1.8 Voltage-Gated Sodium Channel; D062558:NAV1.9 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; C568319:Scn10a protein, rat; C113705:Scn11a protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C023790:tetrodotoxin-binding protein; D013779:Tetrodotoxin; D007811:Lanthanum; D002118:Calcium