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Bi 1 “Drugs and the Brain” Lecture 22 Revised 5/18/06 Monday, May 15, 2006 1. Long-QT syndrome; 2. Epilepsy 3. Hunt

The final in-class slide (but not the posted slides) has a 1-point extra credit question. You may not communicate this question to another student; no collaboration. Bi 1 “Drugs and the Brain” Lecture 22 Revised 5/18/06 Monday, May 15, 2006 1. Long-QT syndrome; 2. Epilepsy

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Bi 1 “Drugs and the Brain” Lecture 22 Revised 5/18/06 Monday, May 15, 2006 1. Long-QT syndrome; 2. Epilepsy 3. Hunt

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  1. The final in-class slide (but not the posted slides) has a 1-point extra credit question. You may not communicate this question to another student; no collaboration. Bi 1 “Drugs and the Brain” Lecture 22 Revised 5/18/06 Monday, May 15, 2006 1. Long-QT syndrome; 2. Epilepsy 3. Huntington’s Disease

  2. Na+ channels conduct K+ channels conduct R T P Q S Action potentials and the electrocardiogram Action Potential measured with intracellular electrode ~ 100 mV ~ 1 sec Electrocardiogram measured on the skin ~ 100 mV

  3. Monday, May 15. 2006 8:52 AM Kaiser Sunset Facility Cardiology Lab, Treadmill facility Part of Bi1 lecturer Bi1 lecturer’s baseline EKG

  4. An approximate explanation for the electrocardiogram, slide 1 The left ventricle pumps against the greatest resistance therefore it has thickest walls; therefore its currents are the largest; therefore it contributes most of the ECG.

  5. The capacitive currents are largest Cl- K+ Na+ An extracellular electrode pair records IR drops proportional to the (absolute value) of the 1st derivative of membrane potential. G C E like Lecture 6 An approximate explanation for the electrocardiogram, slide 2 extracellular Cl- K+ Na+ G C E cytosol

  6. extracellular K+ Cl- Na+ K+ Cl- Na+ G G C C E intracellular E An approximate explanation for the electrocardiogram, slide 3 chest Only a small fraction of the current flows across the resistance between chest and a limb. This produces a DV ~ 103 times smaller than the transmembrane potential. The ECG records this signal leg

  7. Na+ channels conduct K+ channels conduct R T P Q S Action potentials and the electrocardiogram Action Potential measured with intracellular electrode ~ 100 mV ~ 1 sec Electrocardiogram measured on the skin ~ 100 mV

  8. a heart-specific Na channel fails to inactivate completely Or, one of several heart-specific K channels fails to activate Two classes of V-dependent channel explain cardiac electrophysiology in long-QT Syndrome. ~ 8 genes (complementation groups) Action Potential Electrocardiogram R Normal heart rhythm T P Q S Arrhythmia Q-T

  9. A cardiac K channel is also the target for drug-induced arrhythmias Seldane® blocks hERG and was pulled from the market; Allegra® does not

  10. Epilepsies: Repeated Seizures Seizure: Massive derangement of brain function caused by excessive and synchronized function in a group of neurons. A seizure can range from a “focal” 3-sec loss of consciousness, barely noticeable (like a “space out”) . . . to a “generalized” event that causes a person to tense for several sec before a several sec jerking of his entire body. Prevalence: ~ 5% of the general population experiences one or more seizures. The repeated seizures termed epilepsy occur in ~0.5% of the population. Causes: brain injury (included a traumatic blow to the head), chronic illness, and inherited vulnerabilities . Genetics: ~ 50% of epilepsies involve an inherited vulnerability.

  11. Epilepsies caused by Bi 1 Molecules Genetics: ~ 50% of human epilepsies involve an inherited vulnerability. Many knockout mice have seizures. Most of these genes are not associated with human epilepsies. Nestler Table 21-3 lists ion channel defects that produce some inherited epilepsies (also discussed in Problem set 7). KNCQ, a family of K channels (loss of function). SCN, a Na channel (gain of function). CHRN, nicotinic acetylcholine receptors (gain or loss, still uncertain). Problem Set 6, Q1; see next slides. In general, the causal links are less well understood than for long-QT syndrome.

  12. An exemplar inherited epilepsy: Autosomal dominant nocturnal frontal lobe epilepsy. First described as a disease, 1994. The first epilepsy gene mapped and sequenced (1995). Seizures arise during phase 2 sleep (rather than “rapid eye-movement sleep”; Sometimes confused with nightmares. Some patients display abnormal brain waves (as in Nestler Figures 21-5, 21-6). Controlled by carbamazepine, not by valproate

  13. Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005) from Lecture 3: ~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 106 Binding region Membrane region Colored by secondary structure Colored by subunit (chain) Cytosolic region http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9

  14. from Lecture 3: How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view Ligand-binding domain M1 M2 M3 M4

  15. ADNFLE and slow-channel myasthenic syndrome IC loop Ligand-binding domain M1 M2 M3 M4 Brain Autosomal Dominant Nocturnal Frontal Lobe Epilepsy 2' 6' 9' 10' 14' 18' 22' L a4 I T C I S V L L L S L T V F L L L I T X X X L V b2 T C I S V L L A L T V F L L L I S K I V L Slow-Channel Myasthenic Syndrome: Abnormally long channel duration Muscle a1 M T S I S V L L S L T V F L L V I V L b F L L I A L L T V F L L L L A M S G T e F N V I L V L Q T V L F L I A C S T A d S V L I V L Q S V F L L L I S T A S A Aligned Sequences of Mouse Muscle AChR M2 Domains

  16. from Lecture 8: Procaine Blocks Na+ Channels from inside the cell inside “Trapped” or “Use-Dependent” Blocker Functioning channel procaine-H+ procaine-H+ procaine

  17. from Lecture 8: Na+ channel blockers in medicine Local anesthetics Dental surgery (procaine = Novocain®) Sunburn medications Antiarrhythmics (heart) “use-dependent blocker” example: (procainamide) Antiepileptics / anticonvulsants (brain) “use-dependent blocker” (phenytoin = Dilantin®)

  18. ~ 40 Angstroms (4 nm) transmembrane domain based on Lecture 3: Some drugs compete with nicotine or acetylcholine Carbamazepine, an antiepileptic drug, binds in the pore Nicotinic acetylcholine receptor

  19. from Lecture 21 Huntington’s Disease 1. Clinical description 2. Genetics 3. Gene structure 4. Huntingtin as a protein 5. Physiology of huntingtin 6. What’s wrong with the HD protein? Cystic Fibrosis 1. Clinical description 2. Genetics 3. Gene structure 4. CFTR as a protein 5. Physiology of CFTR 6. What’s wrong with DF508? 7. The cholera connection 8. Selective advantage of CF? 9. Therapeutic approaches: Incremental approaches Gene therapy

  20. 1. Clinical description Onset at 30-40 yr. Neurons in the striatum and cerebral cortex die, leading to movement disorders (“chorea”), dementia, and eventually death. Woody Guthrie 1912-1967 Mother died of Huntington’s chorea; Woody began suffering in ~ 1945 He had 8 children.

  21. from several previous lectures “striped” Again, we highlight neurons that make dopamine; here, note their postsynaptic targets in the striatum GABA-producing “medium spiny” neurons die in HD Nestler Figure 8-6

  22. like Lecture 21 Dominance: 50% of offspring have HD heterozygous “carrier” heterozygous “carrier” homozygous WT homozygous WT HD WT WT HD WT WT WT WT HD phenotype HD phenotype normal phenotype normal phenotype 2. Genetics Huntington’s is a rare autosomal dominant disease (1 in 104 - 105 persons). heterozygous mutant parent “carrier” homozygous WT parent HD WT huntingtin WT huntingtin WT huntingtin HD phenotype normal phenotype

  23. 3. Gene structure (from Lecture 20) First localized to 4p16.3 (~ 2.2 Mb) in 1983. Gene product identified in 1993. Personal decision: does a person at risk for HD submit to the decisive test based on DNA sequencing? Mutation 5’ (N-terminus) 3’ (C-terminus) 210 kb in length 67 exons, 3144 amino acids = 9432 nt coding region (~ 4% of the gene)

  24. 4. Huntingtin as a protein A CAG repeat, encoding glutamine, is amplified. When (CAG)n grows beyond n = 42, the disease occurs. As n increases, age of onset decreases. Eight other human neurodegenerative diseases are caused by expanded triplet repeats. A bafflingaspect of these diseases: the proteins are expressed widelyin brain and other tissues, yet each is toxic in a different,highly specific group of neurons and produces a distinct pathology.

  25. 5. Physiology of huntingtin We don’t know the normal function of huntingtin. “Knockout mice” for huntingtin die early in embryonic development, before the nervous system develops. 6. What’s wrong with the mutant huntingtin? Mice expressing mutant huntingtin exhibit a progressive neurologic phenotype with many of the features of HD, including -choreiform movements, -involuntary stereotypic movements, -tremor, and epileptic seizures, -nonmovement disorder components. Evidently the mutant huntingtin has a destructive effect that is not provoked by wild type huntingtin; thus HD is produced by a “gain-of-function” mutation.

  26. Improper protein aggregates in HD An N-terminal fragment of huntingtin containing the polyglutamine stretch accumulates as aggregates in cells. The aggregates often appear in the nucleus. When this fragment is expressed in mice, or even in yeast, the fragment aggregates as well. It is not known whether the fragment is itself toxic, or whether the nuclear localization is important for toxicity. Huntingtin interacts with several other proteins in the cell. aggregate aggregate Nucleus

  27. Drosophila provides insights, as usual fly expressing polyglutamine repeats glutamine repeats plus “chaperone” proteins wild type fly Seymour Benzer found recently that polyglutamine repeats also distort the development of fruit fly eyes. The polyglutamine repeat has been tagged with GFP, and the proteins clearly aggregate Normal development can be “rescued” with “chaperone” proteins, which help to fold or eliminate misfolded proteins. But the aggregates remain, suggesting that the aggregates themselves are not toxic. low-power electron microscope light microscope GFP

  28. Cl- out in N R-domain C CFTR-DF508 from lecture 21 polyglutamine forms b-sheets Misfolded mutant proteins: a postulated common theme in inherited disease

  29. < 10 nm like Lecture 11 (FRET) detects polyglutamine aggregates blue photon yellow photon virtual cyan photon Cyan Fluorescent Protein (CFP) Yellow Fluorescent Protein (YFP)

  30. No interaction, no FRET fused to CFP fused to YFP

  31. fused to CFP fused to YFP Aggregation leads to FRET

  32. A type of fluorescence microscopy: fluorescence recovery after photobleaching (Ataxin is another triple repeat protein) 1. Use a laser to bleach all the GFP-tagged protein within the rectangle 2. Watch unbleached mobile GFP-tagged “short” ataxin (above) diffuse into the square from other regions of the cell But “long” ataxin in aggregates (below) is immobile for many minutes PNAS (2002), 99, 9310

  33. from Lecture 18 Controlled proteolysis takes place in the proteasome Mutant huntingtin may escape proteolysis in proteasomes because (1) there are no proteasomes in the nucleus (2) mutant huntingtin may be in a complex that cannot be degraded modified from Little Alberts 1st edition Fig 7-32

  34. Intracellular inclusions in some neurodegenerative diseases Alzheimer’s Disease Parkinson’s Disease Huntington’s Disease We don’t know whether these aggregates are part of the disease process, Or simply relatively harmless epiphenomema.

  35. Bi 1 “Drugs and the Brain” End of Lecture 22

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