1. Henry Lester June 2009
Engineering Ion Channels for Selective Neuronal Activation and Silencing

2. 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

3. = The “channelohm” is 2% of the human genome,
and many other organisms expand the repertoire
Binding
region
Membrane
Cytosolic
(incomplete)
Colored by
subunit
(chain)
Voltage (actually, ΔE ~107 V/m)
External transmitter
Internal transmitter
Light
Temperature
Force/ stretch/ movement
Blockers
Switches
Resistor
Battery =
1/r = 0.1 – 100 pS
Nernst potential for
Na+,
K+,
Cl-,
Ca2+, H+
Invertebrate glutamate-gated Cl- channel .
At this resolution, resembles nicotinic acetylcholine receptor

4. The drugs: “avermectins”
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
(IVM)

5. IVM-induced silencing in GluCl-expressing cultured rat hippocampal neurons
500 nm IVM
50 nm IVM
5 nm IVM

6. Optimized constructs optGluCla,b = “AVMR-Cl”
Binding site:
subunit unmutated; b Tyr182Phe (cation-π site)
suppresses endogenous glutamate sensitivity
M3-M4 intracellular loop: a YFP; b CFP
allows visualization
Coding region: codons adapted for mammalian expression
~ 10-fold greater expression A B C D

7. 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)

8. M3-M4 loop Plans to extend the AVMR system
Transfer AVM sensitivity to mammalian glycine receptor
 no immune response
Tighter AVM binding
 increased AVM sensitivity
M2 mutations
 increased AVM sensitivity
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
M3-M4 loop

9. Very slow (several hr) AVM reversibility is puzzling
GluCl- heteromer
GluCl- homomer
No potentiation
GluCl- homomer
Glutamate sensitive?
IVM sensitive?
Potentiation of a glutamate response by IVMPO4?
GluCl-
Yes
GluCl-
Yes (---) No
GluCl-
Yes ()
(Etter et al., JBC 1996)

10. 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)
Cys-loop
89 loop
1mM Glu
1mM IVM
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 /min
(Cully & Paress, 1991)

11. The first AVMR-Na Still too small Still too large (200 nM IVM) ND98
GluCl  WT
+  WT
Muscle nAChR
(10 nM IVM)
Still too small
Subunits
Reversal potential α β
ND98
0.5 ND98
Muscle nAChR
-6.2 ± 0.2
-24.6 ± 0.3 WT
-16.2 ± 0.4
-2.8 ± 0.5
aA13’V
-21.7 ± 1.0
-5.3 ± 1.6
A13’V
T290V
-20.3 ± 0.4
-2.7 ± 0.8
P(-2’)D/A(-1’)E
-6.3 ± 1.0
-15.8 ± 1.7
G(-1’)E
--5.5 ± 1.1 *
-20.5 ± 1.8*
-16.6 ± 1.1
-3.3 ± 2.4
GluCl 
P(-2’)/A(-1’)E
+  WT
Still too large
(200 nM IVM)

12. Many AVMRs remain in intracellular compartments, but are chaperoned by IVM
(GluClαYFP)GluClβ
24 h incubation
(control solution)
The intensity ratio, peripheral/whole cell, is 0.86 ± 0.07 in control and 1.51 ± 0.10 in IVM-treated cells (SEM
(1 μM IVM)
Confocal TIRF