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BMB 170c
Prepares You to Contribute
to
Three Neuroscience Problems that Involve Ion Channels
Presenter: Henry Lester 26 May 2009
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
1/45
Superfamilies of Neurotransmitter-gated Ion Channel Receptors
Cys-loop Receptors
Nicotinic ACh
5HT-3
GABAA and GABAC
Glycine
Ionotropic Glutamate
Receptors
AMPA-type
Kainate-type
NMDA-type
ATP (P2X)
Receptors
2/45
The NMDA receptor is blocked by Mg2+
in a voltage-dependent manner
Mg2+
glutamate
outside
Functioning channel
inside
-30 mV or more positive
Mg2+-blocked channel
-60 mV or more negative
3/45
The NMDA receptor conducts only when
1. The membrane potential is more positive than -30 mV
2. Glutamate is present
(intracellular concentrations of glutamate and Mg2+ are nearly irrelevant)
Action potential
plus
glutamate
functioning channel
Na+, Ca2+
-30 mV
outside
inside
A molecular coincidence detector leading to Na+ and Ca2+ influx,
with many intracellular effects
Including long-term potention (LTP)
4/45
Divalent Cations
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:
5/45
H2O
carbonyl
K+ ion
KcsA structure
K+ ions lose their waters of hydration and
are co-ordinated by backbone carbonyl groups
when they travel through a channel.
6/45
As ions pass through ligand-gated channels,
Hydroxyl side chains partially substitute for waters of hydration
Postulated example: Nicotinic receptor
?
7/45
Time required to exchange waters of hydration
Na+ , K+
1 ns
(~ 109/s)
Ca2+
5 ns
(2 x 108/s)
Mg2+
10 ms
(105/s)
Na+ , K+, and Ca2+ can flow through single
channels at rates > 1000-fold greater than Mg2+
As the most charge-dense cation, Mg2+ holds its
waters of hydration most tightly.
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
Molecular lifetimes
Concentration of
high
acetylcholine at
A synapse
(because of
0
acetylcholinesterase,
turnover time
~ 100 μs)
State 1
closed
State 2
k21
all molecules
begin here at
t= 0
open
units: s-1
Number of open
channels
ms
9/45
. . . . the foot-in-the-door scheme
current
time
10/45
Model or
scheme
normal function
simple block
State 1
all molecules
begin here at
t= 0
State 2
k21
open
closed
k21
open
closed
k23 = k+[Drug]
drug
blocked
k23 = k+[Drug]
k21
foot-in-the-door
closed
drug
blocked
open
k32
Not allowed
11/45
n =1
time constant
= 1/k21
0
time constant
= 1/(k21+ k23)
+
etc
12/45
Localizing the V-dependent binding / blocking site for Mg2+ in the NMDA channel
McMenimen KA, ACS Chem Biol., 2006
13/45
BMB 170c
Prepares You to Contribute
to
Three Neuroscience Problems that Involve Ion Channels
Presenter: Henry Lester 26 May 2009
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
14/45
Nearly Complete Cys-loop Receptor (February, 2005)
~ 2200
amino acids
in 5 chains
(“subunits”),
Binding
region
MW
~ 2.5 x 106
Membrane
region
Colored by
secondary
structure
Colored by
subunit
(chain)
Cytosolic
region
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
Ligand-binding
domain
. . . Until 9 Nov 2005
b2 a4
13’Val
9’Leu
S
I
L
L
L
F
V
T
L
A
L
L
V
S
I
C
L
T
T
I 18'
L
L
L
F
V 13'
T
L
S 10'
L 9'
L
V
S 6'
I
C
L
T 2'
M1
M2
M3
M4
Intracellular
loop
Miyazawa, Fujiyoshi, Unwin, Nature16/45
2003
Nicotine and ACh act on many of the same receptors, but . . .
1. Nicotine is highly membrane-permeant. ACh is not.
Ratio unknown, probably > 1000.
4
2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~10 /s.) In
mouse, nicotine is eliminated with a half time of ~ 10 min.
5
Ratio: ~10
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?
17/45
The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)
aY198
C2
aW149
B
aY93
A
aY190
C1
non-aW55
D
(Muscle Nicotinic numbering)
18/45
Nicotine makes a stronger cation-π interaction with Trp B
at α4β2 receptors than at muscle receptors;
this partially explains α4β2 receptors’ high binding affinity for nicotine.
WT
nicotine EC50,mM
1,000
Receptor
muscle (~3-fold)
a4b2
(47-fold)
100
10
1
WT,
without cation-π
interaction
4
3
2
1
0
Number of F-Trp atoms
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
W149
N
H
O
N+
H
H
N
C2
O
T150
N
H
C1
replace i+1 by
analogous
a-hydroxy acid
A
N
W149
O
BO
H
O
N+
H
Tah150
N
H
D
weakened
hydrogen bond
Weaker
hydrogen
bond
Deleted
hydrogen
bond
20/45
Nicotine EC50 values:
Muscle nAChR
α4β2
single component
~ 400 μM
two components
~ 1 μM, ~200 μM
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.
Xiu, Puskar, Shanata, Lester, Dougherty. Nature 2009
21/45
Changes with chronic nicotine
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).
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
Upregulation is a part of SePhaChARNS
Nicotine is a
“Selective Pharmacological Chaperone
of
Acetylcholine Receptor Number
and
Nicotine
Addiction
Parkinson’s
Disease
ADNFLE
Stoichiometry”
Behavior
Circuits
Synapses
Neurons
Intracell.
Binding
Nic vs ACh
Proteins
RNA
Genes
23/45
Thermodynamics of SePhaChARNS
Free
subunits
+
+
Free Energy
#1. Nicotine binds to subunit interfaces,
favoring assembled receptors
Increasingly
stable
assembled
states
Reaction Coordinate
Bound
states
with
increasing
affinity
+
unbound
Free Energy
#2. Binding eventually
favors high-affinity states
Highest affinity
bound state
C
AC
A2C
A2O
Reaction Coordinate
A2D
24/45
Thermodynamics of SePhaChARNS, #3.
Reversible stabilization amplified by covalent bonds?
+ nicotine
RHS
RLS
Degradation
Covalently
stabilized
AR*HS
?
Nicotine
Increased
High-Sensitivity
Receptors
hr
0
20
40
60
26/45
High-resolution fluorescence microscopy to study SePhaChARNS
TIRFM
LTP / Opioids: regulation
starts here
PM
ER
Pharmacological chaperoning:
upregulation starts here
FRET
Golgi
Nucleus
27/45
Förster resonance energy transfer (FRET): a test for subunit proximity
α4
N
M3 - M4
loop
C
β2
N
C
M3-M4 loop
Ligand binding M1 M2 M3
M4
HA tag XFP
c-myc tag XFP
b2-XFP
a4-XFP
FRET pairs
(m = monomeric)
λ→
XFP =
mCerulean
ECFP
mEGFP
EYFP
mEYFP
mVenus
mCherry
2200
2000
b2ECFP
a4EYFP
Number of Pixels
1800
Data: Data1_C54
Model: Gauss
Equation: y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)
Weighting:
y
No weighting
1600
1400
Chi^2/DoF
= 288.49226
R^2
= 0.9912
1200
y0
xc
w
A
1000
800
4.19078
12.03086
20.05847
12986.99416
±1.65095
±0.06603
±0.15945
±114.84783
600
400
200
0
Neuro2a
FRET
NFRET
-20
0
20
40
% FRET Efficiency
60
80
28/45
Theory of FRET in pentameric receptors with αnβ(5-n) subunits
50% α-CFP, 50% α-YFP
a
a
a
a
a
a
1/4
1/2
1/4
E
No FRET
b/a =1.62; 1.62-6 = 0.055
a
a
a
a
a
1/8
1/4
1/8
1/4
a
E1
a
E2
a
a
E3
a
a
a
a
a
1/8
a
a
a
1/8
a
E4
No FRET
100% α3β2
100%
α2β3
100%(a4)3(b2)2
FRET Efficiency
80
60
100%(a4)2(b2)3
40
20
0
0
20
40
60
80
100
Distance between adjacent subunits, A
% receptors with α3
29/45
A key SePhaChARNS experiment:
changes in subunit stoichiometry caused by chronic nicotine
16
Nicotine
Addiction
Parkinson’s
Disease
ADNFLE
Neuro2a
% FRET Efficiency
14
12
Behavior
10
Circuits
8
Synapses
6
Neurons
4
Intracell.
2
Binding
0
Nic vs ACh
control
+ Nicotine
control
+ Nicotine
a4CFP + a4YFP) : b2
a4 b2CFP + b2YFP)
1:1
1:1
Proteins
RNA
Genes
30/45
Differential subcellular localization and dynamics of α4GFP* receptors
plasma memb. mCherry
α4GFPβ2 (1:1)
overlay
α4GFPβ4 (1:1)
overlay
α4GFPβ2
3 RXR/β subunit
α4GFPβ4 (1:1)
zero RXR/β subunit
31/45
BMB 170c
Prepares You to Contribute
to
Three Neuroscience Problems that Involve Ion Channels
Presenter: Henry Lester 26 May 2009
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
32/45
Neuronal Engineering with Cys-loop receptors
Goal: develop a general technique to selectively and reversibly
silence or activate
specific sets of neurons in vivo.
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
33/45
The “channelohm” is 2% of the human genome,
and many other organisms expand the repertoire
Voltage (actually, ΔE ~107 V/m)
External transmitter
Internal transmitter
Light
Temperature
Force/ stretch/ movement
Blockers
Binding
region
Switches
Membrane
region
Colored
by
subunit
(chain)
=
Resistor
1/r = 0.1 – 100 pS
Battery
Cytosolic
region
(incomplete)
Invertebrate glutamate-gated Cl- channel .
At this resolution, resembles nicotinic acetylcholine receptor
Nernst potential for
Na+,
K +,
Cl-,
Ca2+,
H+
34/45
The drugs
“avermectins”
• IVM: Lactone originally isolated from Streptomyces
avermitilis
• AVMs are used as antiparasitics in animals and
(IVM)
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
The channel resembles the nicotinic receptor & requires two subunits
35/45
First tests: HEK cells
36/45
IVM-induced silencing in GluCl-expressing cultured rat hippocampal neurons
500 nm IVM
50 nm IVM
10m V
5 nm IVM
10mV
25s
10mV
2.5s
2.5s
-48mV
-55m V
60
Conductance (nS)
40
40
50
30
30
40
 = 6sec
20
 = 40s
30
20
  ~ 500s
20
10
0
10
10
0
0
50
100
Tim e (s)
150
0
50
100
Time (s)
150
0
0
400
800
Tim e (s)
1200
37/45
Fluorescent Labels in the M3-M4 loop, function is retained
A
a, YFP;
b, CFP
ab
aab
(FRET shows that the subunits co-assemble)
38/45
We wish to eliminate possible
glutamate sensitivity in GluCl
Cation-p sidechain
Aligns with
GluClb Y182
Colored by
subunit
(chain)
39/45
bY182F eliminates glutamate responses but retains IVM responses
1 mm Glu
1 mM IVM
40/45
Excessive variability among culture dishes
Conductance (nS)
80
60
40
20
0
0
IVMPO4 (nM) 5
41/45
Optimized constructs optGluCla,b=“AVMR-Cl”
Binding site:
asubunit unmutated; b Tyr182Phe (cation-π site)
suppresses endogenous glutamate sensitivity
M3-M4 intracellular loop: a YFP; b CFP
allows visualization
A
B
C
D
IVM-induced conductance (nS)
Coding region: codons adapted for mammalian
expression
~ 10-fold greater expression
50
40
30
20
10
0
0.1
1
10
IVM concentration (nM)
100
42/45
AAV-2 constructs injected into mouse striatum; slice experiments
Single neurons: correlation between IVM-induced conductance & AP silencing
43/45
Plans to extend the AVMR system
main immunogenic region
Transfer AVM sensitivity to
mammalian glycine receptor
 no immune response
agonist binding
a
Tighter AVM binding
 increased AVM sensitivity
extra
Pre-M1
M2 mutations
 increased AVM sensitivity
M2-M3
loop
Na+-permeable
 selective neuronal activation
M2
Ca2+-permeable
 manipulate signal transduction
Increased single-channel current
 increased AVM sensitivity
Amphipathic
helix
anesthetic/
dye binding
trans
M1-M2
loop
ion flow
intra
(inco
44/45
Generating the first AVMR-Na
-60
-40
0.6
0.4
0.4
I (mA)
GluCl a WT + b WT
0.5
I (mA)
ND96 ND96
0.5 (ND96
+ Mannitol)
0.5(ND6 + Mannitol)
0.8
ND96
0.5 (ND96 + Mannitol)
Muscle nAChR
0.2
-20
20
-0.2
40
-60
-40
Vm (mV)
0.2
0.1
-20
20
-0.1
40
Em (mV)
-0.2
-0.4
(10 nM IVM)
0.3
-0.3
-0.6
-0.4
-0.8
-0.5
I (mA)
Still too small
GluCl a P304/A305E
+ b WT
0.15
0.10
0.05
-60
-40
-20
-0.05
-0.10
Still too large
(200 nM IVM)
20
Em (mV)
40
Im
Vm
-0.15
-0.20
-0.25
45/45