2. 1 Bi 1 Lecture 9 Thursday, April 13, 2006 Synapses: Biophysical Machines Reminder: Henry Lester’s “office” hours Mon, Thurs 1-1:30 PM, outside the Red Door

3. 2 Video on synaptic transmission

4. 3 Narrated by Leonard Nemoy

5. 4 Cerebral cortex Axons Dendrites Synapses Synaptic vesicles (volleyballs), mitochondria (watermelons) 1. Movements do not occur in 1 ms; vesicles are already “docked”. 2. Neurotransmitter diffuses across the synaptic cleft in a few  s 3. There is little space between neurons 4. “Slight” errors cause mental illness? Unproven! (But single-codon mutations do cause some neuroscience diseases)

6. 5 Nestler Figure 2-2 (rotated) Parts of two neurons synaptic cleft direction of information flow dendrites Excitatory terminal cell body nucleus axon presynaptic terminal postsynaptic dendrite Inhibitory terminal presynaptic terminal neuron Presynaptic neuron Postsynaptic Greek, “tree” Greek, “axis” from Lecture 4

7. 6 presynaptic neuronpostsynaptic neuron The synapse is a point of information processing An adult human brain contains ~ 10 11 neurons, and each of these might receive 10 3 synapses apiece, for a total of 10 14 synapses. Most of these synapses form during the first 2 yr of life. Thus 10 14 synapses/10 8 s = 10 6 synapses/s form in a fetus and infant! Nestler Box 2-3 Figure A Greek, “connection, junction” from Lecture 4

8. 7 cytosol receptor cytosol synaptic cleft receptor Chemistry is a language of the nervous system, for instance at synapses presynaptic terminal postsynaptic dendrite direction of information flow from Lecture 4 transmitter molecules in synaptic vesicles

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10. 9 Electron microscope image (“micrograph”) of a portion of a synapse synaptic vesicles presynaptic terminal postsynaptic dendrite It takes 3 different pump / transporter proteins to position the transmitter within the vesicles

11. 10 Na + -coupled cell membrane neurotransmitter transporters: Antidepressants (“SSRIs” = serotonin-selective reuptake inhibitors): Prozac, Zoloft, Paxil, Celexa, Luvox Drugs of abuse: MDMA Attention-deficit disorder medications: Ritalin, Dexedrine, Adderall, Strattera (?) Drugs of abuse: cocaine amphetamine Na + -coupled cell membrane serotonin transporter Na + -coupled cell membrane dopamine transporter cytosol outside major targets for drugs of therapy and abuse Presynaptic terminals From Lecture 5 Trademarks:

12. 11 from Lecture 5: Could cells utilize plasma membrane H + fluxes? Probably not. There are not enough protons to make a bulk flow, required for robustly maintaining the ion concentration gradients. (but some very small organelles (~ 0.1  m) do indeed store energy as H + gradients).

13. 12 Neurotransmitter and ATP (1,000 to 10,000 molecules of each) 3. Proton-coupled neurotransmitter transporter cytosol 2. ATP-driven proton pump H+H+ cytosol ~ isotonic! How synaptic vesicles fill from the cytosol vesicle interior

14. 13 Neurotransmitter and ATP kinesin cell body presynaptic terminal ~ 20 distinct proteins vesicle transport; pumping protons; pumping neurotransmitter; docking; fusion; recycling. cytosol 50 nm Lecture 10 Synaptic vesicles have many proteins

15. 14 Neurotransmitter and ATP (1,000 to 10,000 molecules of each) proton-coupled vesicular neurotransmitter transporter cytosol ATP-driven proton pump pH effects also account for some drug actions on synaptic vesicles 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”, XTC) pK a ~ 8.5

16. 15 proton-coupled vesicular serotonin transporter cytosol ATP-driven proton pump synaptic cleft Na + -coupled cell membrane serotonin transporter MDMA serotonin vesicle MDMA MDMA-H + H+H+ MDMA (“ecstasy”) dissipates the vesicle’s H + store, preventing the vesicle from pumping serotonin serotonin depleted serotonin vesicle MDMA-H + Weak acids and weak bases short-circuit many vesicles!

17. 16 These proteins have evolved in a natural—perhaps necessary--way to provide that The resting potential arises via selective permeability to K + This selective permeability also leads to the Nernst potential. Transient breakdowns in membrane potential are used as nerve signals. Neuronal and non-neuronal cells also signal via transient influxes of Na + and Ca 2+. 3 classes of proteins that transport ions across membranes: Little Alberts 12-4 © Garland Ion channels that flux many ions per event Ion-coupled transporters “Active” pumps that split ATP from Lecture 5

18. 17 How does the electric field across a biological membrane compare with other electric fields in the modern world? 1.A “high-voltage” transmission line 1 megavolt = 10 6 V. The ceramic insulators have a length of ~ 1 m. The field is ~ 10 6 V/m. 2.A biological membrane The “resting potential” ~ the Nernst potential for K +, 60 mV. The membrane thickness is ~ 3 nm = 30 A. The field is (6 x 10 -2 V) / (3 x 10 -9 m) = 2 x 10 7 V/m! Dielectric breakdown voltages (V/m) Ceramic8 x 10 7 Silicone Rubber3 x 10 7 Polyvinyl chloride7 x 10 6 from Lecture 6: Introduction to Voltage-Gated Channels

19. 18 K + ions lose their waters of hydration and are co-ordinated by backbone carbonyl groups when they travel through a channel. H2OH2O K + ion carbonyl From Lecture 5

20. 19 H2OH2O K + ion carbonyl From Lecture 5 As of 13 April 2006, there are no crystal structures of voltage-gated Na + and Ca 2+ channels. But the similarities in sequence allow us to assume that the secondary and tertiary structures resemble those of K + channels. A voltage-gated Na+ channel can be changed to a voltage-gated Ca2 + channel by mutating... just 2 out of 1800 amino acids

21. 20 docked vesicle nerve impulse voltage-gated Ca 2+ channel neurotransmitter Electricity, then chemistry triggers synaptic vesicle fusion

22. 21 voltage-gated Ca 2+ channel Electricity, then chemistry triggers synaptic vesicle fusion Ca 2+ docked vesicle neurotransmitter nerve impulse

23. 22 fused vesicle Ca 2+ neurotransmitter Electricity, then chemistry triggers synaptic vesicle fusion

24. 23 A conotoxin: 25 amino acids held together by disulfide bonds individual conotoxins specifically block individual ion channels This conotoxin blocks Ca 2+ channels. Slightly modified, it is now the drug, ziconotide. It suppresses transmission at pain synapses in the spinal cord. (Swiss-prot viewer must be installed on your computer) http://www.its.caltech.edu/~lester/Bi-1/conotoxin-annotated.pdb from Lecture 8:

25. 24 V measured postsynaptic response 1 ms 5 mV -60 +60 large “synaptic potential” leads to postsynaptic action potential subthreshold synaptic events (revealed in low Ca 2+ ) stimulus to presynaptic neuron, producing action potential Electrophysiological analysis of quantal synaptic transmission (slide 1) Nestler Box 2-3 Figure A

26. 25 V we stimulate the presynaptic neuron, producing an action potential we measure the postsynaptic responses, “postsynaptic potentials” repeated identical stimuli to the presynaptic neuron...... yield variable postsynaptic responses! 5 mV 5 ms Electrophysiological analysis of quantal synaptic transmission (slide 2)

27. 26 no stimulus; spontaneous “miniature” postsynaptic potentials repeated stimuli to presynaptic neuron 5 mV 50 - 1000 channels (differs among types of synapse). This is the contents of a single vesicle. Electrophysiological analysis of quantal synaptic transmission (slide 3) 0 1 2 3 4 5

28. 27 N vesicles per terminal (3 in this example) p probability of release per vesicle what is the probability P of releasing n vesicles? (n = 2 for this action potential) N and p sometimes change during memory, learning, and drug addiction Electrophysiological analysis of quantal synaptic transmission (slide 4): Binomial statistics of vesicle release

29. 28 1.Stimulated postsynaptic potentials have variable amplitudes 2.Spontaneous “miniature” postsynaptic potentials occur The amplitudes of the stimulated psp’s are integral multiples of the spontaneous “miniature” psp’s Electrophysiological analysis of quantal synaptic transmission (slide 5): Summary of the classical evidence:

30. 29 fused vesicle adds capacitance C E G Na + K+K+ Cl - inside outside CC inside outside A more direct electrical measurement of quantal release: Measuring the presynaptic capacitance increase due to vesicle fusion

31. 30 To measure the conductances, we set I C = CdV/dt = 0, but  G/dt  0. To measure capacitance, we set I C = CdV/dt  0, but  G/dt = 0. C E G Na + K+K+ Cl - CC Measuring the presynaptic capacitance increase due to vesicle fusion  C ~ 1 femtofarad = 1 fF = 10 -15 F like Lecture 6 http://www.srl.caltech.edu/sal/ph1bc/phys1c.html http://www.theory.caltech.edu/people/politzer/web2.html Phys1c reminders. Anal: Practical:

32. 31 The light chain of botulinum toxin is an enzyme that cleaves synaptic vesicle fusion proteins

33. 32 Popular press: http://search.news.yahoo.com/search/news/?p=botox What’s the latest new use for Botox ? Scientific journals: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Details&DB=PubMed

34. 33 Membrane trafficking, fusion and sorting: crucial to many aspects of cell function. © Garland publishing

35. 34 Membrane trafficking, fusion and sorting: crucial to many aspects of cell function. © Garland publishing

36. 35 Date: Thursday, 13 April 2006 11:38:53 -700 To: Henry Lester lester@caltech.edulester@caltech.edu From “Mary B. Kennedy” kennedym@its.caltech.edukennedym@its.caltech.edu Subject: I wish to interrupt Bi 1 Hi Henry, I wish to tell the Bi 1 students about the biochemistry of postsynaptic signalling and regulation. Mary Mary B. Kennedy, 4/14/05, I wish to interrupt Bi 1

37. 36 Email from Professor Mary Kennedy http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&- response=Detail_Person.html&-layout=all_fields&person_id=11610&-search

38. 37 How do synapses Learn? The answer is mostly Biochemistry

39. 38 The NMDA receptor conducts only when 1. The membrane potential is more positive than -30 mV 2. Glutamate is present outside inside Action potential plus glutamate functioning channel Na +, Ca 2+ A molecular coincidence detector leading to Na + and Ca 2+ influx, with many intracellular effects (lectures 12, 14) -30 mV (intracellular concentrations of glutamate and Mg 2+ are nearly irrelevant)

40. 39 Calcium ion is a biochemical trigger (Often called a “Second Messenger”)

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44. 43 Ca 2+ -dependent protein kinase (12 subunits)

45. 44 Protein kinases are enzymes that catalyze transfer of phosphate to hydroxyl groups on proteins. The phosphate moiety is transferred from ATP to serine, threonine, or tyrosine side chains

46. 45 Phosphorylation of synaptic proteins can change the behavior of synapses

47. 46 End of Lecture 9 Reminder: Henry Lester’s “office” hours Mon, Thurs 1-1:30 PM, outside the Red Door

48. 47 Electrical studies on synapses Nestler Box 2-3 Figure A presynaptic neuron postynaptic neuron

49. 48 no stimulus; spontaneous events repeated stimuli to presynaptic neuron 5 mV 50 - 1000 channels (differs among synapses). This responds to the contents of a single vesicle. 0 1 2 3 4 5 Electrophysiological analysis of synaptic transmission (slide 3)