1 Bi 1 Lecture 7 Monday, April 10, 2006 The Central Dogma of Drugs and the Brain, Part 1: Drugs open and block ion channels Nicotine
2 [neurotransmitter] open closed chemical transmission at synapses: electric field open closed electrical transmission in axons: actually, E Major Roles for Ion Channels from Lecture 6
3 nicotine procainebotulinum toxin Atomic-scale Structures (Swiss-prot pdb viewer must be installed on your computer) morphine from Lecture 2
4 nicotinic acetylcholine receptor Most drug receptors are membrane proteins Outside the cell Inside the cell = cytosol (view in ~1995) natural ligand (agonist) nicotine, another agonist Membrane = lipid bilayer ~ 100 Å = 10 nm from Lecture 3
5 Overall topology of the nicotinic acetylcholine receptor (view in ~2000) outside the cell: 5 subunits each subunit has 4 -helices in the membrane (20 membrane helices total) Little Alberts figure © Garland publishing from Lecture 3
6 The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region (Swiss-prot viewer must be installed on your computer) Color by chain Show 2 subunits, Chains, Ribbons 5 subunits Little Alberts figure © Garland publishing 1/AChBP+Carb-5mer.pdb from Lecture 3
7 The AChBP binding site occupied by an acetylcholine analog (2004) from Lecture 3
8 Binding region Membrane region Cytosolic region Colored by secondary structure Colored by subunit (chain) Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005) ~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 10 6 from Lecture 3
9 How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view M2 M1 M3 M4 Ligand-binding domain from Lecture 3
10 5 kinked -helices rotate, removing the oily side chain ( = hydrophobic) from the pore and exposing side chains with -OH groups. This would provide a water-like (HOH) environment for the permeating ions. One hypothesis about the nature of nicotinic receptor gating in the transmembrane domain
11 Twisting the knee, or straightening the leg? We don’t yet know Engineering & Science, 2002
12 A better way: record the current from channels directly? A Feynman’s idea from Lecture 6
13 5 pA = 10 4 ions/ms 20 ms a nicotinic acetylcholine receptor exposed to acetylcholine A sensitive electronic ammeter dynamic range: 5 s to 5 min 1 part in 10 8 Implementing Feynman’s Idea
14 How ”tight” is the gigaohm seal? 1. Electrically tight See next slide Little Alberts 12-22A © Garland 1 m
15 How ”tight” is the gigaohm seal? 1. Electrically tight R = l/A R ~ 10 9 = resistivity = 22 -cm; l = length = 10 m; A = area = 10 m x t (thickness); Therefore t ~ 2 x m, or less than 1 Å!
16 acetylcholine in the pipette opens channels in the pipette 2. Chemically tight The seal compartmentalizes molecules. Molecules outside the pipette do not mix with molecules inside the pipette acetylcholine outside the pipette opens channels outside the pipette How ”tight” is the gigaohm seal?
17 Alberts omitted all of the electronics! A sensitive electronic ammeter Little Alberts 12-22D © Garland
18 Max Delbruck Richard Feynman H. A. L Carver Mead “If you want to measure small, noisy signals, I have a Senior who can help” from Lecture 6
19 Fred Sigworth ‘74 and Apostol’s Clock 1c-An/index.html#text Ma 1c-Analytic track, Spring 2006: bs/faculty/sig_fr.html Fred Sigworth’s Web page at Yale
20 Statistical analysis of single-molecule events channel opens now we synchronize artificially on the opening event n =1 0
21 Statistical analysis of single-molecule events n =1 0
22 from Chem 1b 2006 Lecture Series #5 (Heath)
23 Concentration of acetylcholine at a synapse Number of open channels ms 0 high closed open State 1State 2 k 21 all molecules begin here at t= 0 units: s -1
24 We wish to test a hypothesis that a particular side chain governs channel opening/closing rates
25 Site-Directed Mutagenesis: the General Idea RNA Gene (DNA) measure Hypothesis about an important side chain(s) Mutate the desired codon(s) “Express” the protein with an altered side chain(s)
26 in vitro RNA synthesis RNA polymerase promoter DNA measure Site-Directed Mutagenesis on Ion Channels Express by injecting into immature frog eggs Mutate the desired codon(s) Latin, ‘in glass” measure
27 The identified side chain governs channel opening/closing rates n =1 0 0 leucine (wild type) alanine
28 We wish to test a hypothesis that a particular side chain governs agonist binding
29 Measured “dose-response” relations verify that an identified side chain governs agonist-receptor interactions wild type (tryptophan) phenylalanine The instrument (~ 90 MB!): p?docnum=475&prodid=108&useid=25&familyid=&interestid=
30 The cation- site in a nicotinic acetylcholine receptor We know that acetylcholine and nicotine bind within a “box” of aromatic amino acids; Electrophysiology agrees with crystallography! ~ 8 Angstroms tryptophan
31 Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms Quantum-mechanical calculations of cation- energy Measured acetylcholine binding energy Dougherty group, Caltech Chemistry
32 End of Lecture 7 Nicotine