ZOO4670/5670 Lecture No. 6. Neurotoxins as Tools for Studying Ion Channel Structure and Functions. Univ. Wyoming. Today s outline

Transcription

1 1 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

2 2 Apamin Honey bee Blocks Ca2+ activated potassium channels Honey bee Stichodactyla Toxin Sea Anemone Blocks voltage-gated potassium channels sea anemone

3 3

4 4 Palytoxin Soft coral Activates sodium channels Capsaicin Cayenne Pepper Excites peripheral nerve endings

5 5 Batrachotoxin Poison Arrow Frog Prevents sodium channels from closing Maculotoxin Blue-Ringed Octopus Blocks sodium channels

6 6 Model or scheme normal function State 1 State 2 closed k 21 open all molecules begin here at t= 0 units: s -1 units: M -1 s -1 k 23 = k + [Drug] simple block closed k 21 open drug blocked current time constant = 1/k 21 time constant = 1/(k 21 + k 23 ) none Ligand binding and dose response curve Concentration of toxin (nm)

7 7 use-dependent block= open channel blocker inside Functioning channel Antiarrhythmics (heart) use-dependent blocker example: (procainamide) Trapped or Use-Dependent Blocker Antiepileptics / anticonvulsants (brain) use-dependent blocker (phenytoin = Dilantin ) A use-dependent blocker stimuli impulses (voltage) impulses fail channel population (currents) pronounced block at brief intervals threshold little block at long intervals

8 8 Green mamba snake Pore region sequence of ShaKv1.1. The dendrotoxin receptor surface of ShaKv1.1. KcsA The surface color represents the change in binding energy of dendrotoxin associated with mutation

9 9 Conclusions: dendrotoxin binds near the pore entryway but does not act as a physical plug. Hypothetical binding orientation for dendrotoxin Scorpion toxin fits even better to the pore entryway! Binding orientation of scorpion toxin on the K. channel.

10 10 C11H17N3O8 Tetrodotoxin beta-scorpion toxins alpha-scorpion toxin Sea anemone Motif III b-scorpion toxins trap IIS4 in the outward, activated position. Channel must be activated for toxin effect. Kd =0.1nM a-scorpion toxin trap IVS4 in an inward, partially activated position. Prolonged depolarization drives the toxins off their receptor site Motif I Motif IV

11 11 Neurotoxins that block Na+ channels Local Anesthetics Etidocaine Lidocaine Many others Antiarrhythmic Drugs Lidocaine Mexiletine Quinidine Flecainide Anticonvulsants Phenytoin Carbamazepine Lamotrigine use dependent block

12 12 Baldomero Toto Olivera Ph D olivera@biology.utah.edu Distinguished Professor Olivera lab directory Geographer s cone Magician s cone Leopard cone Cloth-of-gold cone

13 A conotoxin: 27 amino acids held together by disulfide bonds individual conotoxins specifically block individual ion channels. 13

14 14 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 Conus magus, 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 ziconotide appeared 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.

15 15 Cerebral cortex (layer II/III) Thalamus Trigeminal nucleus

16 16 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. Toxins that act on voltage-gated ion channels Name Source Neuronal Action Agatoxin Funnel Web Spider Blocks calcium channels Agitoxin Scorpion Blocks potassium channels Apamin Honey bee Blocks potassium channels Atracotoxin Blue Mountains Funnel Web Blocks voltage-gated calcium channels Spider 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 Grammotoxin SIA 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

17 17 Toxins that act on voltage-gated ion channels: continued 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 Stichodactyla Toxin Taicatoxin Tetrodotoxin (TTX) Tityustoxin-K Versutoxin Sea Anemone Australian Taipan snake Pufferfish Brazilian Scorpion Funnel web spider Blocks voltage-gated potassium channels Inhibits voltage-gated calcium channels Blocks sodium channels Blocks potassium channels Opens sodium channels