Come a little closer the venom will hurt but you will Live,

maybe

…

Njeri Henderson Kristin Wright Damon Morgan Phoenix Tantum Biology of Toxins 445 April 17 th 2013

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Why should we care?

Background of Black Widow Spider (B.W.S.) Characteristics of B.W.S.

Introduction to α-Latrotoxin (α-LTX) Mechanism of Action Deeper Investigations of α-LTX Symptoms Treatment Toxicity Levels Antivenin Medical Uses Interesting Facts Summary

 Before you can understand the Toxin, it is useful to explore the Animal which is responsible for it’s mechanism  There is an irrational fear of Black Widow Spiders and exploring their Toxicity will hopefully alleviate this assumption  In humans α-latrotoxin RARELY result in death  α-latrotoxin has POTENTIAL MEDICAL USES

Arachnophobia: An irrational fear of spiders and is considered among the most common of all phobias Of the 30,000 types of spiders, the black widow is one of the best known and feared

Most widespread venomous land animal, ranging from the Mediterranean, across the Americas, all of Africa and much of Australia and in Russia and Asia. • • genus:

Latrodectus mactans

mixture of Latin and Greek : species latro - robber / macto - to slay translation: “deadly biting robber”

Adult Black Widow Juveniles have more potent venom (more neurotoxins) Their venom is 15x more potent than the Prairie Rattlesnake Juvenile Black Widow

Females sometimes kill the male after mating, hence their name “Black Widow” Adults reach a maximum size of half an inch Shiny, black, globular abdomen with a distinctive red hourglass on the underside Their venom contains latrotoxin, a protein which attacks both the nervous system and the hormones which regulates how the body functions The vertebrate-specific neurotoxin is α-latrotoxin (α-LTX)

 Selective for vertebrates - ineffective in insects and crustaceans  High affinity for receptors that are specific for neuronal and endocrine cells  Alpha latrotoxin consists of four sub units an α-LTX dimer and δ-LIT dimer that form a tetramer. IT IS ONLY TOXIC IN TETRAMERIC FORM  In order to for the tetramer to form there must be cations such as Ca 2+ present.

In its tetrameric form it interacts with receptors on the neuronal membrane which causes the insertion of α-LTX into the membrane, where the action of the venom takes place

 This tetramer then binds to membrane receptors neurexin I, latrophilin 1, and receptor-like protein tyrosine phosphatase σ (PTP σ) found on neuronal membranes of motor cells and endocrine cells.  Once the tetramer is inserted into the cell membrane two mechanisms of actions can occur: ◦ 1. Pore Formation ◦ 2. Receptor-mediated effects

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Pore Formation: Pores are permeable to Ca2+

Pores are also permeable to neurotransmitters, cations and water Influx of Ca2+ into the cell Stimulates exocytosis Nerve terminal swelling This stimulates a massive release of neurotransmitters resulting in clonic contractions of muscle tissue.

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Receptor-mediated effects:

α-LTX stimulates a receptor (latrophilin 1-G protein receptor) Linked to Gαq/11 Phospholipase C (PLC) is the downstream effector of Gαq/11 Induces release of Ca2+ from intracellular stores When PLC is activated it increases cytosolic concentration of IP3 o Latrophilin with α-LTX can also induce the activation of Protein Kinase C (PKC).

Increased rate of spontaneous exocytosis o PKC is responsible for the phosphorylation of SNARE proteins.

o Thus latrophilin with α-LTX induces the effect of exocytosis of transport vesicles.

o This Mechanism is not yet known.

 α-LTX’s ability to form pores in plasma membrane has been used to study mammalian nervous systems.

 Helped confirm the vesicular hypothesis of transmitter release.  Establish the requirement of Ca 2+ for endocytosis.

 Characterized the existence of individual neurotransmitter release sites within central nervous system.

 Mutant form of α-LTX, α-LTX N4C , does not form pores in membrane.

 Stimulation of neuronal receptors by α-LTX N4C intracellular Ca 2+ stores. mediates release of  Excitatory stimulation caused by intracellular Ca endocytosis of additional Ca 2+ The readily releasable pool, and the depot pool. 2+ release without indicates two pools of synaptic vesicles:

 The Bite ( in: IS THE ONLY WAY FOR THE TOXIN TO BE ABSORBED VIA THE BLOOD ) is similar to a pin prick, which takes its effect in 30 to 60 minutes which results ◦ Pain in bite are may become generalized ◦ Muscle cramps (especially shoulder and back) ◦ Abdominal pain – may mimic conditions of appendicitis or gallbladder problems ◦ Chest pain – may be mistaken for a heart attack ◦ Blood Pressure and Heart Rate Elevated ◦ Nausea, vomiting, fainting, dizziness and respiratory problems may follow People RARELY die from a black widow's bite. Life-threatening reactions are generally seen only in small children and the elderly.

 The victim usually recovers in 2 to 5 days. Only 1% of all black widow attacks are fatal.

 Despite the low mortality rates it is still recommended that one immediately see a Doctor after an bite.  Calcium gluconate (anti-venom) can also be administered by an physician  The wound should also be cleansed and Ice Packs can be used for the pain

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The LD

50

of α-LTX measured in μg/kg:

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Guinea Pig: 0.1

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Pigeon: 0.4

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Mouse: 0.9

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Chicken: 2.1

Human LD50 ranges from 4.3 to 20 to 95 μg/kg

 Find spider  Milk spider  Purify venom  Inject pure venom into horse or sheep  Animal makes antibodies against venom  Withdraw blood and separate antibodies  Concentrate antibodies into a solution  Inject into blood of recently bitten victim

 Indigenous South Americans have traditionally used the venom as an Aphrodisiac  Chilean scientists are working on a peptide derived from the venom to produce a Male Contraceptive Agent (Romero et. al., 2011)  The venom Counters The Effects Of innvervation of muscles paralyzed by Botulinum Toxin (Gomez et. al., 1981)  A particular peptide with a building block of proteins could help Treat Alzheimer's’

Could be First Documented Death from α-LTX No deaths have been reported in the past 10 years in the U.S.

 Daniela Holmqvist, a rookie from Sweden on the Ladies European Tour was bitten by a spider during a round in Canberra, Australia.

Most recent case of α-LTX in humans February 14, 2013

 Black Widow spiders, regions. Latrodectus mactans , are ubiquitous in nearly all  Black Widow venom is a tetramer polypeptide that is injected into the spiders prey.  Once in the bloodstream α-LTX binds to neuronal and endocrine cell membranes, inducing pore formation and Ca extreme cases death. 2+ uptake, which in turn causes vesicular neurotransmitter exocytosis. This produces clinical symptoms such as muscle pain, tachycardia, diaphoresis, and in  Most Black Widow envenomation's do not result in long term health problems, and death is rare .

 α-LTX has been used to research neuronal cell function, and uses for Black Widow venom as a therapeutic agent for several conditions are being investigated.

               Davletov, Bazbek A. Frederic A. Meunier. Anothony C. Ashton. Hiroaki Matsushita. Warren D. Hirst. Vera G. Lelianova. Graham P. Wilkin. J. Oliver Dolly. And Yuri A. Ushkaryov. External and AStored Ca 2+ .

From: Vesicle Exocytosis Stimulated by Alpha-Latrotoxin is Mediated by Latrophilin and Requires Both The EMBO Journal. Vol. 17 No.14. pp.3909-3920. 1998.

Garb, Jessica E., and Cheryl Y. Hayashi. Widow Spider Venom.

Molecular Evolution of Alpha-Latrotoxin, the Exceptionally Potent Vertebrate Neurotoxin in Black http://mbe.oxfordjournals.org/ Obtained on: March 23, 2013 Holts, George G. and Joel F. Habener. Black Widow Spider Alpha-Latrotoxin: A Presynaptic Neurotoxin that Shares Structural Homology with the Glucagon-like Peptide-1 Family of Insulin Secretagogic Hormones. From: Comp Biochem Physiol B Biochem Mol Biol.

1998 October; 121(2):177-184 Ichtchenko, Konstantin. Mikhail Khvotchev. Nikita Kiyatkin. Lance Simpson. Shuzo Sugita. And Thomas C. Südhof. Alpha-Latrotoxin Action Probed with Recombinant Toxin: Receptors Recruit Alpha-Latrotoxin but do not Transduce an Exocytotic Signal. From: The EMBO Journal.

Vol. 17 No.21 pp. 6188-6199. 1998 Khvotchev, Mikhail. And Thomas C. Südhof. Plasma Membrane.

From: The EMBO Journal. Alpha-Latrotoxin Triggers Transmitter Release via Direct Insertion into the Presynaptic Vol. 19 No.13. pp. 3250-3262. 2000 Silva, Amelia M., June Liu-Gentry, Adam S. Dickey, David W. Barnett, and Stanley Misler. Depolarization-Evoked Exocytosis from Pancreatic Islet Beta-Cells.

From: Alpha-Latrotoxin increases spontaneous and Journal of Physiology . Vol. 565, Issue 3. Pp. 783-799. 2005.

Silva, John-Paul, Jason Suckling, and Yuri Ushkaryov. Penelope’s Web: Using Alpha-Latrotoxin to Untangle Mysteries of Exocytosis.

Journal of Nuerochemistry . 2009 October; 111(2): 275-290. Doi:10.1111/j.4159.2009.06329.x.

From: Trudeau, Louis-Eric. Vladmir Parpura. And Philip G. Haydon. During Recovery From Intracellular Acidification.

From: Activation of Nuerotransmitter Release in Hippocampal Nerve Terminals Journal of Neurophysiology. 81:2627-2635. 1999 Tzeng, Mu-Chin. Rochelle S. Cohen. And Philip Siekevitz. Release of Neurotransmitters and Depletion of Synaptic Vesicles in Cerebral Cortex Slices by Alpha-Latrotoxin From Black Widow Spider Venom.

From: Neurobiology. Vol. 75 No.8. pp.4016-4020. August 1978.

Ushkaryov, Yuri A., Alexis Rohou. And Shuzo Sugita. Alpha-Latrotoxin and its Receptors.

2008; (184): 171-206. Doi: 10.1007/978-3—540-74805-2_7 From: Handbook Experimental Phamacology.

Utah Poison Control Center. Black Widow Spider Envenomation.

From: Utox pdate.

Vol. 4, Issue 3. 2002 B. Ceccarelli, W.P. Hurlbut, Ca 2+ -dependent recycling of synaptic vesicles at the frog neuromuscular junction, J. Cell Biol., 87 (1980), pp. 297–303 C. Auger, A. Marty, Heterogeneity of functional synaptic parameters among single release sites, Neuron, 19 (1997), pp. 139–150 A.C. Ashton, K.E. Volynski, V.G. Lelianova, E.V. Orlova, C. Van Renterghem, M. Canepari, M. Seagar, Y.A. Ushkaryov, α-latrotoxin, acting via two Ca 2+ -dependent pathways, triggers exocytosis of two pools of synaptic vesicles , J. Biol. Chem., 276 (2001), pp. 44695–44703