Image retrieved from Lyndsay Esson Connor Gibney Samantha Nail Dylan Radke PHM142 Fall 2015 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson

What Is A Sodium Channel ?  Integral membrane protein  Large alpha subunit which contains 4 domains. Each domain consists of 6 transmembrane regions  Allows the movement of Na + ions across cell membranes Blumenthal, K. M., Seibert, A. L. (2003). Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochemistry and Biophysics, 38 (2), Image retrieved from:

Role In Physiology  Crucial in propagation of action potentials  CLOSED at rest  OPENED during depolarization  INACTIVATED during repolarization Costill, D. L., Kenney, W. L., Wilmore, J. H. (2011). Physiology of sport and exercise: 5 th edition. Human Kinetics Images retrieved from: Stanfield, C. S., Germann, W. J.(2007). Principles of human physiology. Benjamin Cummings.

Functional Sites on Sodium Channel  Site 1: Binds toxins that physically occlude the pore  Site 2: Binds toxins on cytoplasmic and intramembranous regions causing persistent activation  Site 3: Toxin binding causes increased open probability  Site 4: Binding causes a shift of channel activation to more hyperpolarized values. Blumenthal, K. M., Seibert, A. L. (2003). Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochemistry and Biophysics, 38 (2),

Tetrodotoxin (TTX)  Also known as fugu toxin  One of the most common sodium channel poisons Can be found in several species including the california newt, pufferfish, starfish and angel fish  Poisoning by TTX most commonly occurs through consumption of improperly prepared pufferfish

The TTX binding site  TTX is a site-1 toxin of the sodium channel Blocks the channel and prevents sodium ions from crossing the membrane, blocking neurotransmission  Binding site is extracellular for TTX  Positive charges of TTX coordinate with highly conserved negative residues on the sodium channel which are organize into two rings around the poor, between S5 and S6 Inner ring residues: Glu-387, Glu 945, Asp-1426, Asp-1717 Outer ring residues: Asp-384, Glu-942, Lys-1422, Ala-1714 Blumenthal, K.M, Seibert, A.L., (2003) Voltage-gated sodium channel toxins. Cell Biochemistry and Biophysics 38: Terlau, H., Heinemain, S.H., Stuhmer, W., Pusch, M., Conti, F., Imoto, K., Numa, S., (1991) Mapping the site of block by tetrdotoxin and saxitoxin of sodium channel II. Federation of European Biochemical Societies. 1(2):93-96

Symptoms of TTX poisoning  TTX poisoning occurs in 2 phases following ingestion Phase 1: numbness of the mouth begins about 20 minutes after ingestion, followed by loss of sensation in the face and limbs Phase 2: Paralysis begins to set in and movement becomes difficult and the victims ability to breath is compromised. Symptoms progress until respiratory and cardiac function cease and the victim dies, approximately 8 hours after ingestions depending on the dose U.S. FDA, Centre for Food Safety and Applied Nutrition. Tetrodotoxin, Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. Accessed November 10 th 2015

Batrachotoxin (BTX)  Isolated from the Poison Dart Frog  Potent Cardiotoxic and Neurotoxic Steroidal Alkaloid  Irreversibly binds to the sodium channels and forces them to remain open  Causes muscle contractions, violent convulsions, fibrillation, arrhythmias, heart failure, and death.

A Model of the Binding Li, H-L., Hadid, D., Ragsdale, D. (2002) The Batrachotoxin Receptor on the Voltage-Gated Sodium Channel is Guarded by the Channel Activation Gate. Mol Pharmacol. 61:

Method of Action  Modifies the Sodium Channel in Five Ways 1. Shifts the Threshold for Action Potentials Closer to Depolarization by mV 2. No Fast Inactivation of Na+ Channels 3. No Slow Inactivation of Na+ Channels 4. Reduces Single Channel Conductance 5. Alters Ion Selectivity Honerjager, P., Reiter, M. (1977) The Cardiotoxic Effect of Batrachotoxin. Naunyn-Schmiedeber’s Archives of Pharmacology. 299(3):

Management of Sodium Channel Poisoning  Tricyclic antidepressants often cause cardiac toxicity via sodium channel blockage Ie. Flecainide, Imiprine  Hypertonic sodium salts most widely used treatment for this cardiac toxicity Sodium bicarbonate or lactate Di Grande, A., et al. "Management of sodium-channel blocker poisoning: the role of hypertonic sodium salts." Eur Rev Med PharmacolSci 14 (2010):

Mechanisms of Action Hypertonic salts work by sodium loading and acting as alkalinizing agents Thought to antagonize sodium channel blockage by; ○ increasing the unbinding rate ○ Electrostatic repulsion ○ interacting with the inactivated state intracellularly to decrease concentrations of the drug * Mechanism and therapeutic efficacy still poorly understood Bou-Abboud, Elias, and Stanley Nattel. "Molecular mechanisms of the reversal of imipramine-induced sodium channel blockade by alkalinization in human cardiac myocytes." Cardiovascular research 38.2 (1998): Di Grande, A., et al. "Management of sodium-channel blocker poisoning: the role of hypertonic sodium salts." Eur Rev Med PharmacolSci 14 (2010):

Therapeutic Benefits of TTX Use of TTX as an Analgesic agent  Defined role in relieving normal and neuropathic pain Via interference of action potential generation in nervous system  Has been tested against cancer pain clinically  Potential role in acute and inflammatory pain Further clinical research still required Nieto, Francisco Rafael, et al. "Tetrodotoxin (TTX) as a therapeutic agent for pain." Marine drugs 10.2 (2012):

Summary Slide  Hetero-oligomeric protein; 4 domains that each contain 6 transmembrane regions  Role in the human body is propagation of action potentials  TTX is a site 1 sodium channel poison which prevents neurotransmission Positively charged TTX binds to negative residues of aspartic and glutamic acid on the extracellular pore of the sodium channel Causes increasing paralysis as the toxin moves through the body until cardiac depression causes death  BTX is a site 2 sodium channel poison which prevents the sodium channel from closing. Modifies the Sodium Channel is 5 different ways.  Some sodium channel poisoning can be treated with hypertonic sodium salts  TTX can be used therapeutically for pain relief

References  Blumenthal, K. M., Seibert, A. L. (2003). Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochemistry and Biophysics, 38 (2),  Bou-Abboud, Elias, and Stanley Nattel. "Molecular mechanisms of the reversal of imipramine-induced sodium channel blockade by alkalinization in human cardiac myocytes." Cardiovascular research 38.2 (1998):  Costill, D. L., Kenney, W. L., Wilmore, J. H. (2011). Physiology of sport and exercise: 5 th edition. Human Kinetics  Di Grande, A., et al. "Management of sodium-channel blocker poisoning: the role of hypertonic sodium salts." Eur Rev Med PharmacolSci 14 (2010):  Li, H-L., Hadid, D., Ragsdale, D. (2002) The Batrachotoxin Receptor on the Voltage- Gated Sodium Channel is Guarded by the Channel Activation Gate. Mol Pharmacol. 61:  Honerjager, P., Reiter, M. (1977) The Cardiotoxic Effect of Batrachotoxin. Naunyn- Schmiedeber’s Archives of Pharmacology. 299(3):  Nieto, Francisco Rafael, et al. "Tetrodotoxin (TTX) as a therapeutic agent for pain." Marine drugs 10.2 (2012):  Terlau, H., Heinemain, S.H., Stuhmer, W., Pusch, M., Conti, F., Imoto, K., Numa, S., (1991) Mapping the site of block by tetrdotoxin and saxitoxin of sodium channel II. Federation of European Biochemical Societies. 1(2):93-96  U.S. FDA, Centre for Food Safety and Applied Nutrition. Tetrodotoxin, Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. Accessed November 10 th 2015