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ZOO4670/5670 Lecture No. 6

Neurotoxins as Tools for Studying Ion Channel Structure and Functions

Qian-Quan Sun Ph.D (neuron@uwyo.edu)

Univ. Wyoming

Today’s outline

• 1. Basics about channel block• 2. Snake toxins and potassium channels• 3. Sodium channels: toxins and local

anesthetics• 4. Sea snails, spiders and calcium

channels

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Honey bee

Apamin Honey bee Blocks Ca2+ activated potassium channels

Stichodactyla Toxin Sea Anemone Blocks voltage-gated potassium channels

sea anemone

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Palytoxin Soft coral Activates sodium channels

Capsaicin Cayenne Pepper Excites peripheral nerve endings

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Batrachotoxin Poison Arrow Frog Prevents sodium channels from closing

Maculotoxin Blue-Ringed Octopus Blocks sodium channels

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closed open

State 1 State 2

normal functionk21

all molecules begin here at

t= 0

Model orscheme

closed opendrug

blockedsimple block

k21

k23 = k+[Drug]

units: s-1 units: M-1s-1

time constant= 1/k21

current

none

time constant= 1/(k21+ k23)

Ligand binding and dose response curveConcentration of toxin (nM)

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Functioning channel“Trapped” or

“Use-Dependent” Blocker

inside

use-dependent block= open channel blocker

Antiarrhythmics (heart) “use-dependent blocker”example: (procainamide)

Antiepileptics / anticonvulsants (brain) “use-dependent blocker”(phenytoin = Dilantin® )

pronounced blockat brief intervals

little blockat long intervals

impulses(voltage)

channelpopulation(currents)

stimuli

impulses fail

A use-dependent blocker

threshold

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Green mamba snake

The dendrotoxin receptor surface of ShaKv1.1.

Pore region sequence of ShaKv1.1.

KcsA

The surface color represents the change in binding energy of dendrotoxin associated with mutation

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Conclusions: dendrotoxin binds near the pore entryway but does not act as a physical plug.

Hypothetical binding orientation for dendrotoxin

Binding orientation of scorpion toxin on the K. channel.

Scorpion toxin fits even better to the pore entryway!

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TetrodotoxinC11H17N3O8

Motif IV

Motif I

Motif III

beta-scorpion toxins

b-scorpion toxins trap IIS4 in the outward, activated position.Channel must be activated for toxin effect.

alpha-scorpion toxinSea anemone

a-scorpion toxin trap IVS4 in an inward, partially activated position. Prolonged depolarization drives the toxins off their receptor site

Kd =0.1nM

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Neurotoxins that block Na+ channels

AntiarrhythmicDrugs

LidocaineMexiletineQuinidineFlecainide

AnticonvulsantsPhenytoin

CarbamazepineLamotrigine

Local AnestheticsEtidocaineLidocaine

Many others

use dependent block

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Baldomero “Toto” Olivera Ph D olivera@biology.utah.eduDistinguished ProfessorOlivera lab directory

Magician’s coneGeographer’s cone

Leopard cone Cloth-of-gold cone

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A conotoxin:27 amino acidsheld together by disulfide bondsindividual conotoxins specifically block individual ion channels.

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The lethal sea snail toxin is a promising pain killer .

Ziconotide is the synthetic equivalent of a 25-amino-acid, polybasic peptide from the venom of Conusmagus, a marine snail. It is the first selective N-type, voltage-sensitive calcium-channel blocking agent to reach clinical trials.In the current study, patients aged 24 to 85 years with refractory pain from advanced cancer or AIDS were randomized to ziconotide or placebo in a 2:1 ratio. Intrathecal ziconotide was titrated over 5 to 6 days, followed by a 5-day maintenance phase. Mean pain scores improved 52.9% in the ziconotide group, compared with 17.5% in the placebo group, with no reduction in efficacy during the maintenance phase. Five patients receiving ziconotide achieved complete pain relief.Ziconotide also seems to improve the sleep pattern in some patients with chronic severe pain. Patients experienced positive effects on nocturnal sleep duration and believed that remaining pain symptoms interfered less with their daily life.In experimental studies of absorption, distribution, metabolism, and elimination, intrathecal ziconotideappeared and diminished rapidly in plasma and resulted in relatively little plasma protein binding. Intravenous ziconotide degraded in animal brain tissue in 2 to 24 hours, produced no detectable intermediates, and was cleared quickly from both cerebrospinal fluid (CSF) and the circulatory system. This accelerated clearance rate likely means that the distribution of ziconotide throughout CSF and metabolism within CSF are also rapid.

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Trigeminal nucleus

Cerebral cortex (layer II/III)

Thalamus

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Voodoo• Ancient haitian traditions used the puffer fish for its voodoo

practices. – The tetrodotoxin is used to create zombies– It puts the people in a death-like state

• The towns people would take some to be put into this state

– While under this state, they would be forced to do odd jobs.- The zombies were used as a social damper to scare towns people to keep them in line.

Name Source Neuronal Action

Agatoxin Funnel Web Spider Blocks calcium channels

Agitoxin Scorpion Blocks potassium channels

Apamin Honey bee Blocks potassium channels

AtracotoxinBlue Mountains

Funnel Web Spider

Blocks voltage-gated calcium channels

Batrachotoxin Poison Arrow Frog Prevents sodium channels from closing

Capsaicin Cayenne Pepper Excites peripheral nerve endings

Charybdotoxin Scorpion Blocks potassium channels

Ciguatoxin Dinoflagellate Opens sodium channels

Conotoxin Marine Snail voltage-sensitive calcium channels; sodium channels

Dendrotoxin Green Mamba Blocks voltage-gated potassium channels

GrammotoxinSIA

South American Rose Tarantula

Blocks calcium channels

Gonyautoxin Dinoflagellate Blocks sodium channels

Iberiotoxin Scorpion Blocks potassium channels

Kaliotoxin Scorpion Blocks potassium channels

Kurtoxin South African Scorpion Blocks calcium channels

Toxins that act on voltage-gated ion channels

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Maculotoxin Blue-Ringed Octopus Blocks sodium channels

Margatoxin Scorpion Blocks potassium channels

Noxiustoxin Scorpion Blocks sodium channels

Palytoxin Soft coral Activates sodium channels

Phoneutriatoxin Banana spider Slows sodium channel inactivation

Phrixotoxin Chilean fire tarantula Blocks potassium channels

Robustotoxin Funnel web spider Opens sodium channels

Saxitoxin Dinoflagellate Blocks sodium channels

SNX-482 African Tarantula Blocks calcium channels

StichodactylaToxin Sea Anemone Blocks voltage-gated potassium

channels

Taicatoxin Australian Taipansnake Inhibits voltage-gated calcium channels

Tetrodotoxin(TTX) Pufferfish Blocks sodium channels

Tityustoxin-K Brazilian Scorpion Blocks potassium channels

Versutoxin Funnel web spider Opens sodium channels

Toxins that act on voltage-gated ion channels: continued