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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
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