Download Lester-McKnight-2009

Henry Lester
June 2009
Engineering Ion Channels for Selective Neuronal Activation and Silencing
1/14
Neuronal Engineering with Cys-loop Receptor Channels
Goal: develop a general technique to selectively and reversibly
silence or activate
specific sets of neurons in vivo.
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 or activate “diffuse” molecularly defined sets of neurons, not just spatially
defined groups
The chosen channel
Cys-loop receptor (like nicotinic receptors)
Heteropentamer: α2β3 or α3β2 subunits.
This feature allows one to intersect two promoters, to enhance cellular specificity
2/14
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+
3/14
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
4/14
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
5/14
Optimized constructs optGluCl,b=“AVMR-Cl”
Binding site:
 subunit unmutated; b Tyr182Phe (cation-π site)
suppresses endogenous glutamate sensitivity
M3-M4 intracellular loop:  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
8/14
AAV-2 constructs injected into mouse striatum; slice experiments
Single neurons: correlation between IVM-induced conductance & AP silencing
Lerchner et al, 2007 (collaboration with D. J. Anderson at Caltech)
9/14
Plans to extend the AVMR system
main immunogenic region
Transfer AVM sensitivity to
mammalian glycine receptor
 no immune response
agonist binding

Tighter AVM binding
 increased AVM sensitivity
extra
Pre-M1
M2 mutations
 increased AVM sensitivity
M2-M3
loop
Na+-permeable
 selective neuronal activation
Ca2+-permeable
 manipulate signal transduction
Increased single-channel current
 increased AVM sensitivity
Optimize ER exit and trafficking
→ increased surface expression
M2
Amphipathic
helix
anesthetic/
dye binding
trans
M1-M2
loop
ion flow
M3-M4 loop
intra
(inco
10/14
Very slow (several hr) AVM reversibility is puzzling
GluCl-b
heteromer
GluCl-b
homomer
No potentiation
GluCl-
homomer
Glutamate
sensitive?
IVM
sensitive?
Potentiation of a glutamate
response by IVMPO4?
GluCl-b
Yes
Yes
Yes
GluCl-b
Yes (---)
No
No
GluCl-
No
Yes
Yes ()
11/75
(Etter et al., JBC 1996)
Location of the AVM binding site is unknown
Likely distinct from the glutamate binding site
Within the cavity of the TMD?
At the ECD-TMD interface?
Covalent binding interaction?
(where other anesthetics are bind)
1mM Glu
1mM IVM
Cys-loop
b8b9 loop
McCammon Lab, UCSD
Yoav Paas, BIU
Radioligand binding experiments with [3H]-IVM on C. elegans membrane preps
IVM binding sites exhibit high affinity binding (KD = 0.11 nM)
IVM does dissociate from its receptor, with a rate constant of 0.005-0.006/min
12/75
(Cully & Paress, 1991)
The first AVMR-Na
0.8
-60
I (A)
-40
-60
-40
-60
40
-40
-20
Vm (mV)
I (A)
-20
0.3
0.2
-0.4
-0.3
-0.6
-0.4
0.15
20
-0.1
-0.2
40
Em (mV)
-0.5
-0.8
GluCl 
P(-2’)/A(-1’)E
+ b WT
0.4
0.1
20
-0.2
(10 nM IVM)
ND96
0.5Muscle
(ND96 +nAChR
Mannitol)
0.2
-20
I (A)
ND96
0.6
0.5(ND6 + Mannitol)
0.4
GluCl  WT
+ b WT
0.5
ND98
0.5 ND98
Subunits
Still too small
Reversal potential
α
β
ND98
0.5 ND98
-6.2 ± 0.2
-24.6 ± 0.3
0.10
Muscle nAChR
0.05
WT
WT
-16.2 ± 0.4
-2.8 ± 0.5
A13’V
WT
-21.7 ± 1.0
-5.3 ± 1.6
A13’V
T290V
-20.3 ± 0.4
-2.7 ± 0.8
P(-2’)/A(-1’)E
WT
-6.3 ± 1.0
-15.8 ± 1.7
P(-2’)/A(-1’)E
G(-1’)E
--5.5 ± 1.1 *
-20.5 ± 1.8*
WT
G(-1’)E
-16.6 ± 1.1
-3.3 ± 2.4
-0.05
-0.10
20
Em (mV)
40
-0.15
-0.20
Still too large
-0.25
(200 nM IVM)
13/14
Many AVMRs remain in intracellular compartments, but are chaperoned by IVM
(GluClαYFP)GluClβ
24 h incubation
(control solution)
(1 μM IVM)
The intensity ratio,
peripheral/whole cell, is 0.86 ±
0.07 in control and 1.51 ± 0.10 in
IVM-treated cells (SEM
Confocal
TIRF
14/14