Learning & memory:Detailed pharmacology of Mg block .

Molecular & formal descriptions

Nicotine addiction:Cation-π interactions at the nicotine receptor binding site;

Selective Chaperoning of nicotine receptors

Epilepsy:Engineering Ion Channels

BMB 170c

Prepares You to Contribute

to

Three Neuroscience Problems that Involve Ion Channels

Presenter: Henry Lester 26 May 2009

1/45

Superfamilies of Neurotransmitter-gated Ion Channel Receptors

Cys-loop ReceptorsNicotinic ACh 5HT-3 GABAA and GABAC

Glycine

Ionotropic Glutamate ReceptorsAMPA-typeKainate-typeNMDA-type

ATP (P2X) Receptors

2/45

Functioning channel

Mg2+-blocked channel

The NMDA receptor is blocked by Mg2+ in a voltage-dependent manner

outside

inside-30 mV or more positive

-60 mV or more negative

Mg2+ glutamate

3/45

The NMDA receptor conducts only when 1. The membrane potential is more positive than -30 mV2. Glutamate is present

outside

inside

Action potential plus glutamate functioning channel

Na+, Ca2+

A molecular coincidence detector leading to Na+ and Ca2+ influx,with many intracellular effects

Including long-term potention (LTP)

-30 mV

(intracellular concentrations of glutamate and Mg2+ are nearly irrelevant)

4/45

What is the selective advantage that cells maintain Ca2+ at such low levels?

Cells made a commitment, more than a billion yr ago, to use high-energy

phosphate bonds for energy storage.

Therefore cells contain a high internal phosphate concentration.

But Ca phosphate is insoluble near neutral pH.

Therefore cells cannot have appreciable concentration of Ca2+;

they typically maintain Ca2+ at < 10 –8 M.

What is the selective advantage that cells don’t use Mg2+ fluxes?

The answer derives from considering the atomic-scale structure of a K+ -

selective channel (next slide), which received the 2003 Nobel Chemistry Prize:

Divalent Cations

5/45

K+ ions lose their waters of hydration and

are co-ordinated by backbone carbonyl groups

when they travel through a channel.

H2O K+ ion

carbonyl

KcsA structure

6/45

As ions pass through ligand-gated channels,Hydroxyl side chains partially substitute for waters of hydration

Postulated example: Nicotinic receptor

~60o

closedclosed

-OH

openopen

-OH

-OH

-OH

-OH

-OH

?

7/45

Na+ , K+1 ns

(~ 109/s) Na+ , K+, and Ca2+ can flow through single channels at rates > 1000-fold greater than Mg2+

Ca2+5 ns

(2 x 108/s)

Mg2+

10 s

(105/s)As the most charge-dense cation, Mg2+ holds its waters of hydration most tightly.

Time required to exchange waters of hydration

The “surface / volume” principle:

We know of several Mg2 transporters,

but Mg2+ channels apparently exist only in mitochondria & bacteria.

Moomaw & Maguire, Physiologist, 2008 8/45

Concentration of acetylcholine at

A synapse(because of

acetylcholinesterase,turnover time

~ 100 μs)

Number of open channels

ms

0

high closed open

State 1 State 2

k21

all molecules begin here at

t= 0

units: s-1

Molecular lifetimes

9/45

current

time

. . . . the foot-in-the-door scheme

10/45

closed open

State 1 State 2

normal functionk21

closed opendrug

blockedsimple block

k21

closed opendrug

blockedfoot-in-the-doork21

k32

all molecules begin here at

t= 0

Model orscheme

Not allowed

k23 = k+[Drug]

k23 = k+[Drug]

11/45

time constant= 1/(k21+ k23)

time constant= 1/k21

closed open

State 1 State 2

normal functionk21

closed opendrug

blockedsimple block

k21

closed opendrug

blockedfoot-in-the-door

k21

k32

all molecules begin here at

t= 0

Not allowed

k23 = k+[Drug]

k23 = k+[Drug]

etc

n =1

0

+

12/45

Localizing the V-dependent binding / blocking site for Mg2+ in the NMDA channel

McMenimen KA, ACS Chem Biol., 2006

13/45

Learning & memory:Detailed pharmacology of Mg block .

Molecular & formal descriptions

Nicotine addiction:Cation-π interactions at the nicotine receptor binding site;

Selective Chaperoning of nicotine receptors

Epilepsy:Engineering Ion Channels

BMB 170c

Prepares You to Contribute

to

Three Neuroscience Problems that Involve Ion Channels

Presenter: Henry Lester 26 May 2009

14/45

Binding region

Membrane region

Cytosolicregion

Colored by secondary

structure

Colored by subunit(chain)

Nearly Complete Cys-loop Receptor (February, 2005)

~ 2200 amino acids in 5 chains

(“subunits”),

MW ~ 2.5 x 106

15/45

The 9’Leucine and 13’Valine residues are conserved among most / all Cys-loop receptor subunits and

reside at or near the gate

Miyazawa, Fujiyoshi, Unwin, Nature 2003

13’Val9’Leu M2

M1

M3

M4

T

Ligand-bindingdomain

Intracellular loop

2'

6'

10'

13'

18'

9'

CI

VLL

LTVFLLLI

TL

L

T

CI

VLLALTVFLLLIS

L

S S

S

. . . Until 9 Nov 2005

16/45

1. Nicotine is highly membrane-permeant. ACh is not.

Ratio unknown, probably > 1000.

2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~104 /s.) In

mouse, nicotine is eliminated with a half time of ~ 10 min.

Ratio: ~105

3. EC50

at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM.

Ratio, ~10. Justified to square this because nH = 2. Functional ratio, ~100.

For nicotine, EC50

(muscle) / EC50

(α4β2) = 400

What causes this difference?

Nicotine and ACh act on many of the same receptors, but . . .

17/45

The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)

W149B

Y93A

non-W55D

Y198C2

Y190C1

(Muscle Nicotinic numbering) 18/45

Nicotine makes a stronger cation-π interaction with Trp Bat α4β2 receptors than at muscle receptors;

this partially explains α4β2 receptors’ high binding affinity for nicotine.

4 3 2 1 01

10

100

1,000

nic

otin

e E

C50

,M

Number of F-Trp atoms

Receptor muscle (~3-fold) (47-fold)

WT

WT, without cation-π

interaction

19/45

Nicotine makes a stronger H-bond to a backbone carbonyl

at α4β2 than at muscle receptors:With amide to ester substitution,

EC50 increases 20-fold vs 1.5-fold

NH

HN

NH

W149

O T150

O

replace i+1 byanalogous

-hydroxy acid

NH

ONH

W149

O Tah150

O

HN+ H

N+ weakenedhydrogen bond

A BC1

C2

D

Weaker hydrogen bond

Deleted hydrogen bond

20/45

Xiu, Puskar, Shanata, Lester, Dougherty. Nature 2009

Nicotine EC50 values:

Underlying the 400-fold higher nicotine sensitivity

of

neuronal vs muscle receptors:

Factor of ~16 for the cation-π interaction;

Factor of ~ 12 for H-bond;

16 x 12 = 192. We still can’t explain a factor of 400/192 ~ 2.

Muscle nAChR single component ~ 400 μM

α4β2 two components ~ 1 μM, ~200 μM

21/45

Chronic exposure to nicotine causes upregulation of nicotinic receptor binding

(1983: Marks & Collins; Schwartz and Kellar);

Upregulation 1) Involves no change in receptor mRNA level;

2) Depends on subunit composition (Lindstrom, Kellar, Perry).

Changes with chronic nicotine

Shown in experiments on clonal cell lines

transfected with nAChR subunits:

Nicotine seems to act as a

“pharmacological chaperone” (Lukas, Lindstrom)

or

“maturational enhancer”

(Sallette, Changeux, & Corringer; Heinemann)

or

“Novel slow stabilizer” (Green).

Upregulation is “cell autonomous” and “receptor

autonomous” (Henry). 22/45

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

Upregulation is a part of SePhaChARNS

Nicotine is a

“Selective Pharmacological Chaperone

of

Acetylcholine Receptor Number

and

Stoichiometry”NicotineAddiction

NicotineAddiction

Parkinson’s Disease

Parkinson’s Disease

ADNFLEADNFLE

23/45

+ +

Fre

e E

nerg

y

Reaction Coordinate

Free subunits

Increasingly stable

assembled states

#1. Nicotine binds to subunit interfaces, favoring assembled receptors

+Boundstates with

increasing affinity

Fre

e E

nerg

y

Reaction Coordinate

C

AC

A2C A2O

A2D

Highest affinity bound state

unbound

#2. Binding eventually favors high-affinity states

Thermodynamics of SePhaChARNS

24/45

Thermodynamics of SePhaChARNS, #3.

Reversible stabilization amplified by covalent bonds?

Nicotine

hr0 20 40 60

Increased High-Sensitivity

Receptors

RLS RHS

Covalently stabilized

AR*HSDegradation

+ nicotine

?

26/45

Nucleus

TIRFM

FRET

High-resolution fluorescence microscopy to study SePhaChARNS

Golgi

ER

PMLTP / Opioids: regulation starts here

Pharmacological chaperoning: upregulation starts here

27/45

Förster resonance energy transfer (FRET): a test for subunit proximity

ECFPXFP =EYFP

mEYFPmVenusmCerulean mEGFP mCherry

Ligand binding M1 M2 M3 M4M3-M4 loop

M4

M3 - M4loop

α4

c-myc tag XFP

4-XFP 2-XFP

HA tag XFP

FRET pairs(m = monomeric)

N C N Cβ2

-20 0 20 40 60 800

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Data: Data1_C54Model: GaussEquation: y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)Weighting: y No weighting Chi^2/DoF = 288.49226R^2 = 0.9912 y0 4.19078 ±1.65095xc 12.03086 ±0.06603w 20.05847 ±0.15945A 12986.99416 ±114.84783

Nu

mb

er o

f P

ixel

s

% FRET Efficiency

2ECFP 4EYFP

FRET NFRET

Neuro2a

λ→

28/45

Theory of FRET in pentameric receptors with αnβ(5-n) subunits

No FRET

No FRETE

1/2 1/4 1/4

E1 E2 E3 E4

1/8

1/4

1/4

1/8 1/8 1/8

50% α-CFP, 50% α-YFP

b/a =1.62; 1.62-6 = 0.055

0

20

40

60

80

0 20 40 60 80 100Distance between adjacent subunits, A

FR

ET

Eff

icie

ncy 100%()3()

100%()()3

100% α3β2100% α2β3

% receptors with α3 29/45

A key SePhaChARNS experiment: changes in subunit stoichiometry caused by chronic nicotine

0

2

4

6

8

10

12

14

16

(4CFP + YFP) : 21:1

+ Nicotine

% F

RE

T E

ffic

ienc

y

control

(CFP + YFP)1:1

+ Nicotinecontrol

Neuro2a

NicotineAddictionNicotine

AddictionParkinson’s

DiseaseParkinson’s

DiseaseADNFLEADNFLE

BehaviorBehavior

CircuitsCircuits

SynapsesSynapses

NeuronsNeurons

Intracell.Intracell.

BindingBinding

Nic vs AChNic vs ACh

ProteinsProteins

RNARNA

GenesGenes

30/45

Differential subcellular localization and dynamics of α4GFP* receptors

α4GFPβ2

α4GFPβ4 (1:1)

α4GFPβ2 (1:1) overlayplasma memb. mCherry

α4GFPβ4 (1:1) overlay

3 RXR/β subunit

zero RXR/β subunit

31/45

Learning & memory:Detailed pharmacology of Mg block

Molecular & formal descriptions

Nicotine addiction:Cation-π interactions at the nicotine receptor binding site;

Selective Chaperoning of nicotine receptors

Epilepsy:Engineering Ion Channels

BMB 170c

Prepares You to Contribute

to

Three Neuroscience Problems that Involve Ion Channels

Presenter: Henry Lester 26 May 2009

32/45

Neuronal Engineering with Cys-loop receptors

Rationale: Investigate functional roles of defined neurons in ways not feasible with present techniques.

Therapy for diseases of excessive neuronal activity, e g epilepsy

Ideal approach would:

Have on- and off- kinetics on a time scale of minutes

Have simple activation (ie, via drug injected or in animal’s diet)

Avoid nonspecific effects in animal

Maintain target neurons healthy in an “off-state” for a few days without morphological/other changes

Silence “diffuse” molecularly defined sets of neurons, not just spatially defined groups

Goal: develop a general technique to selectively and reversibly

silence or activate

specific sets of neurons in vivo.

33/45

Binding region

Membrane region

Cytosolicregion(incomplete)

Colored by

subunit(chain)

The “channelohm” is 2% of the human genome,

Voltage (actually, ΔE ~107 V/m)External transmitterInternal transmitter

LightTemperature

Force/ stretch/ movementBlockers

Nernst potential forNa+,

K+,Cl-,

Ca2+,H+

Switches

Resistor

Battery

=1/r = 0.1 – 100 pS

and many other organisms expand the repertoire

Invertebrate glutamate-gated Cl- channel .At this resolution, resembles nicotinic acetylcholine receptor

34/45

The drugs“avermectins”

(IVM)

• IVM: Lactone originally isolated from Streptomyces

avermitilis

• AVMs are used as antiparasitics in animals and

humans (“River blindness” / Heartgard™)

• IVM is probably an allosteric activator of GluCl

channels

•Also modulates GABA, 5HT3, P2X, and nicotinic

channels, at much higher doses

O

O

O

OO

O

O

O

OO

O

OO

O

H

H

H H

H

H

HH

H

HH HH

H

H

HH

H

HH H

H

HH

H

O

O

O

O

OO

O

ON

H

H

H

H

H

H

H

HH

H

H

H H

H

ivermectin B1a

moxidectin

The channel resembles the nicotinic receptor & requires two subunits 35/45

First tests: HEK cells

36/45

-48mV

10mV

2.5s

-55mV

10mV

2.5s

0 50 100 1500

10

20

30

40

50

60

= 40s

Time (s)0 50 100 150

0

10

20

30

40

= 6sec

Co

nd

uc

tan

ce

(n

S)

Time (s)

10mV25s

0 400 800 12000

10

20

30

40

~ 500s

Time (s)

IVM-induced silencing in GluCl-expressing cultured rat hippocampal neurons

5 nm IVM500 nm IVM 50 nm IVM

37/45

Fluorescent Labels in the M3-M4 loop, function is retained

(FRET shows that the subunits co-assemble)

, YFP; , CFP

A

38/45

Colored by subunit(chain)

We wish to eliminate possible glutamate sensitivity in GluCl

Cation-sidechainAligns with GluCl Y182

39/45

Y182F eliminates glutamate responses but retains IVM responses

1 mm Glu 1 M IVM

40/45

0

20

40

60

80

50

Con

duct

ance

(nS

)

IVMPO4 (nM)

Excessive variability among culture dishes

41/45

Binding site: subunit unmutated; Tyr182Phe (cation-π site)suppresses endogenous glutamate sensitivity

M3-M4 intracellular loop: YFP; CFPallows visualization

Coding region: codons adapted for mammalian expression~ 10-fold greater expression

Optimized constructs optGluCl,“AVMR-Cl”

A B

C D

0.1 1 10 1000

10

20

30

40

50

IVM

-indu

ced

cond

ucta

nce

(nS

)

IVM concentration (nM)42/45

AAV-2 constructs injected into mouse striatum; slice experimentsSingle neurons: correlation between IVM-induced conductance & AP silencing

43/45

main immunogenic region

anesthetic/dye binding

intracellular(incomplete)

transmembrane

extracellular

ion flow

agonist binding

M2

M2-M3loop

M1-M2loop

Amphi-pathichelix

Pre-M1

Tighter AVM binding increased AVM sensitivity

Increased single-channel current increased AVM sensitivity

Na+-permeable selective neuronal activation

Ca2+-permeable manipulate signal transduction

Transfer AVM sensitivity to mammalian glycine receptor no immune response

Plans to extend the AVMR system

M2 mutations increased AVM sensitivity

44/45

(200 nM IVM)

GluCl WT + WT

-60 -40 -20 20 40

-0.25

-0.20

-0.15

-0.10

-0.05

0.05

0.10

0.15

I (

A)

Em (mV)

-60 -40 -20 20 40

-0.5

-0.4

-0.3

-0.2

-0.1

0.1

0.2

0.3

0.4

0.5

I (

A)

Em (mV)

ND96 0.5 (ND96 + Mannitol)

GluCl P304/A305E + WT

(10 nM IVM)

-60 -40 -20 20 40

-0.8

-0.6

-0.4

-0.2

0.2

0.4

0.6

0.8

I (

A)

Vm (mV)

ND960.5(ND6 + Mannitol)

Muscle nAChR

Generating the first AVMR-Na

Still too large

Still too small

Im Vm

45/45