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.

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

N

CH3N

Nicotine

2

[neurotransmitter]

openclosedchemical transmission atsynapses:

electric field

openclosedelectrical transmission inaxons:

actually, E

Major Roles for Ion Channelsfrom Lecture 6

3

nicotine procaine

N

CH3N

O

CH2

H2C

NH2

C O

NH3CH2C CH2CH3

botulinum toxin

Atomic-scale Structures

(Swiss-prot pdb viewer must be installed on your computer)

N

O

HO

HOCH3

morphine

morphine

http://www.its.caltech.edu/~lester/Bi-1/nicotine.pdb

http://www.its.caltech.edu/~lester/Bi-1/procaine.pdb

http://www.its.caltech.edu/~lester/Bi-1/morphine.pdb

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 subunitseach subunit has 4 -helices

in the membrane (20 membrane helices total)

Little Alberts figure 12-42© 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 chainShow 2 subunits,Chains,Ribbons

5 subunits

Little Alberts figure 12-42© Garland publishing

http://www.its.caltech.edu/~lester/Bi-1/AChBP+Carb-5mer.pdb

from Lecture 3

7

http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb

The AChBP binding site occupied by an acetylcholine analog (2004)http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb

from Lecture 3

8

Binding region

Membrane region

Cytosolicregion

Colored by secondary

structure

Colored by subunit(chain)

Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)

http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9

~ 2200 amino acids in 5 chains

(“subunits”),

MW ~ 2.5 x 106

from Lecture 3

9

How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view

M2

M1

M3

M4

Ligand-bindingdomain

from Lecture 3

10

~60o

closedclosed

-OH

openopen

-OH

-OH

-OH

-OH

-OH

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 = 104 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 108

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 ~ 109

= resistivity = 22 -cm;

l = length = 10 m;

A = area = 10 m x t (thickness);

Therefore

t ~ 2 x 10-11 m, or less than 1 Å!

t

pipette wall

membrane

t

pipette wall

membrane

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

http://www.math.caltech.edu/classes/ma1c-An/index.html#text

Ma 1c-Analytic track, Spring 2006:

http://www.info.med.yale.edu/bbs/faculty/sig_fr.html

Fred Sigworth’s Web page at Yale

20

Statistical analysis of single-molecule eventschannel 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 1 State 2

k21

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

~60o

closedclosed

-OH

openopen

-OH

-OH

-OH

-OH

-OH

25

Site-Directed Mutagenesis: the General Idea

RNA

Gene (DNA)

measure

Hypothesis about an important side chain(s)

Mutate the desired codon(s)

“Express” theprotein with an altered side chain(s)

26

in vitro RNA synthesisRNA 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

n =1

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!):

http://www.moleculardevices.com/product_literature/download_form.php?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

Mea

sure

d ac

etyl

chol

ine

bind

ing

ener

gy

Dougherty group, Caltech Chemistry

32

End of Lecture 7

N

CH3N

Nicotine