targeting presynaptic cav2€¦ · 2.2 peptides are inhibitory agents • synthetic ca v 2.2...
Post on 20-Aug-2020
1 Views
Preview:
TRANSCRIPT
© University of Reading 2006 www.reading.ac.uk
Gary Stephens
School of Pharmacy, University of
Reading, UK
Targeting presynaptic CaV2.2
calcium channels
1
2 Kamp et al. (2005) EJN 21:1617–1625
Presynaptic Ca2+ channels define cell excitability
Ca2+ channels are heteromultimeric proteins
Catterall (2000) Annu Rev Cell Dev Biol 16:521-55 3
CaVa1
Ca2+ channels are modulatable proteins due to the
presence of intracellular terminals and loops……
4
I
IV III II
N C
Jarvis & Zamponi (2007) Curr Opin Cell Biology 9:474-82
5 Adapted from Jarvis & Zamponi (2007) Curr Opin Cell Biology 9:474-82
PKC PKC RGS12 ●
CRMP2●
Ca2+ channels are modulatable proteins due to the
presence of intracellular terminals and loops……
6
CaV2 subunits are subject to inhibitory Gbg-mediated
modulation
+100 mV +10 mV
CaV2.2/b2a
+Gbg
10 pA/pF
50 ms
+PP
CaV2x
g
d a2
GPI
Ca2+
N
C +Gbg
Nt-I-II loop interaction proposed by Agler et al. (2005) Neuron 46:891-904
I II
7
Question: Which intracellular sites are involved in
transmitter release?
= NT peptide (rat CaV2.2[45-55]): YKQSIAQRART
= AID peptide (rat CaV2.2[377-393]): RQQQIERELNGYLEWIF
CaV2.2
g
d a2
GPI
Ca2+
N
C
a2
NA
presynapse
Gbg I II
Functional studies in rat superior cervical ganglion
neuron (SCGN) model synapses
Collaborator: Prof Sumiko Mochida, Tokyo Medical University
pre
post
10 m
vesicles
Active zone
0.5 μm
Peptide injection
EPSP
AP
8
9
C scrambled AID peptide
A 1 mM AID peptide
No
rma
lise
d E
PS
P
am
plit
ude
Time after injection (m)
-20 -10 0 10 20 30 40 50 60 70 0.2
0.4
0.6
0.8
1.0
1.2
1.4
No
rma
lise
d E
PS
P
am
plit
ude
Time after injection (m)
-20 -10 0 10 20 30 40 50 60 70 0.2
0.4
0.6
0.8
1.0
1.2
1.4
D
De
cre
ase
in
EP
SP
am
plit
ude
(%
)
50
40
30
20
10
0
B 100 M AID peptide
No
rma
lise
d E
PS
P
am
plit
ude
Time after injection (m)
-20 -10 0 10 20 30 40 50 60 70 0.2
0.4
0.6
0.8
1.0
1.2
1.4
control
50 ms
10 mV
AID peptide 20’
Synthetic CaV2.2 peptides inhibit synaptic transmission
in SCGN synapses: AID peptide
1 mM CaV2.2 NT peptide
No
rma
lise
d E
PS
P a
mp
litu
de
Time after injection (m)
- 2 0 0 2 0 4 0 6 0 8 0 1 0 0 0 . 2
0 . 4
0 . 6
0 . 8
1 . 0
1 . 2
1 . 4
-50
-40
-30
-20
-10
0
† D
EP
SP
am
plit
ud
e (
%)
Synthetic CaV2.2 peptides inhibit synaptic transmission
in SCGN synapses: NT peptide
11
Synthetic CaV2.2 peptides inhibit G protein modulation
in SCGN synapses
A B
-20 0 20 40 600.2
0.4
0.6
0.8
1.0
1.2
1.4
No
rma
lise
d E
PS
P
am
plit
ude
Time after NA application (m)
AID peptide
10 M NA C D
D E
PS
P a
mp
litud
e (
%)
-20 0 20 40 600.2
0.4
0.6
0.8
1.0
1.2
1.4
No
rma
lise
d E
PS
P
am
plit
ude
Time after NA application (m)
10 M NA
-20 0 20 40 600.2
0.4
0.6
0.8
1.0
1.2
1.4
No
rma
lise
d E
PS
P
am
plit
ude
Time after NA application (m)
NT peptide
10 M NA
control
-50
-40
-30
-20
-10
0
12
Functional patch clamp studies in isolated rat SCGNs
- 6 0 - 4 0 - 2 0 0 2 0 4 0 6 0
- 1 8
- 1 6
- 1 4
- 1 2
- 1 0
- 8
- 6
- 4
- 2
0
pA
/pF
V (mV)
-90 mV
5 pA/pF
50 ms
-30 mV
-10 mV
+10 mV SCG neuron
extracellular solution:
160 mM TEABr + 10 mM Ba2+
+ 10 M nifepedine:
pure Cav2.2 population
intracellular solution:
140 mM CsAspartate
Ca2+ channel
13
-60 -40 -20 20 40 60
-50
-40
-30
-20
-10
0
10
(pA
/pF
)
V (mV)
control 1 (n=9)
AID peptide
0
5
10
15
20
cu
rren
t d
en
sit
y (
pA
/pF
)
control (n=11)
NT peptide (n=11)
AID peptide (n=10)
Synthetic CaV2.2 peptides inhibit Ca2+ current in
isolated SCGNs
14
Synthetic CaV2.2 peptides inhibit G protein modulation
in isolated SCGNs
A
0
10
20
30
40
50
60
70
NA
% In
hib
itio
n (
at
-10 m
V)
control (n = 11)
NT peptide (n = 11)
AID peptide (n = 10)
AID peptide NT peptide
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
NA
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
NA
Im
Vm
-200 pA
-10 mV
10.00 ms
control
Im
Vm
-200 pA
-10 mV
10.00 ms
NA
Im
Vm
-200 pA
-10 mV
10.00 ms
400
pA
10 ms
-60 -40 -20 0 20 40 600.0
0.2
0.4
0.6
0.8
1.0
No
rm
alis
ed
Ta
il C
urre
nt
V (mV)
Control + PP (n = 10)
Control + NA (n = 10)
-60 -40 -20 0 20 40 600.0
0.2
0.4
0.6
0.8
1.0
NT + NA (n = 11)No
rm
alised
Tail C
urren
t
V (mV)
NT + PP (n = 11)
-60 -40 -20 0 20 40 600.0
0.2
0.4
0.6
0.8
1.0
No
rm
ali
sed
Ta
il C
urre
nt
V (mV)
AID + PP (n = 8)
AID + NA (n = 8)
B
15
CaV2.2 peptides stabilise inhibitory channel conformations
CaV2.2
g
d a2
GPI
Ca2+
N
C
ACh
ACh
ACh
ACh
nACh
R
postsynapse
presynapse
NT peptide AID peptide
I II
16
NT + AID peptide D
Aii Ai NT + AID peptide
400 pA
10 ms
NA
0
10
20
30
40
50
60
70
NA
% in
hib
itio
n (
at
-10 m
V)
control (n = 10)
NT + AID (n = 10)
-60 -40 -20 0 20 40 60
0.0
0.2
0.4
0.6
0.8
1.0
NT + AID + NA (n = 10)
No
rmalised
tail c
urr
en
t
V (mV)
NT + AID + NA + PP
-20 0 20 40 60
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Norm
aliz
ed
EP
SP
am
p
Time after injection (m)
Co-application of AID and NT peptide NEGATES
inhibitory effects
Site 1:
CaV2.2 amino terminal (NT) peptides
NT peptide (rat CaV2.2[45-55]): YKQSIAQRART
CaV2.2 NT R52A,R54A peptide: YKQSIAQAAAT
Site 2:
CaV2.2 I-II loop alpha-interaction domain (AID) peptide
AID peptide (rat CaV2.2[377-393]): RQQQIERELNGYLEWIF
CaV2.2 AID W391A peptide: RQQQIERELNGYLEAIF
17
Further characterization of inhibitory CaV2.2 peptides:
use of mutated peptides
18
NT R52A,R54A peptide C
-20 0 20 40 60 80
0.2
0.4
0.6
0.8
1.0
1.2
1.4
N
orm
alis
ed E
PS
P a
mp
Time after injection (m)
-60 -40 -20 0 20 40 600.0
0.2
0.4
0.6
0.8
1.0
No
rm
ali
sed
Ta
il C
urren
t
V (mV)
mut NT + PP (n = 8)
mut NT + NA (n = 8)
Aii Ai NT R52A,R54A peptide
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
Im
Vm
-200 pA
-10 mV
10.00 ms
NA 400 pA
10 ms
B
0
10
20
30
40
50
60
70
NA
% In
hib
itio
n (
at
-10 m
V)
Control (n = 11)
mut NT (n = 9)
NT (n = 11)
†
CaV2.2 NT R52A,R54A peptide LACKS inhibitory effects
19
CaV2.2 W391A peptide has INCREASED inhibitory effects
-40
-30
-20
-10
0AID peptide
beta mut AID peptide
scrambled AID peptide
D E
PS
P a
mplit
ude
*
*
-20 0 20 40 600.4
0.6
0.8
1.0
1.2
1.4
AID W-A peptide (n=9 )
No
rma
lise
d E
PS
P a
mp
litu
de
Time after bath application ( min)
AID peptide (n=7)
B A
20
-60 -40 -20 20 40 60
-50
-40
-30
-20
-10
0
10
pA
/pF
V (mV)
AID W-A peptide
AID peptide (n=9)
-60 -40 -20 20 40 60
-50
-40
-30
-20
-10
0
10
(pA
/pF
)
V (mV)
control group 2 (n = 9)
AID W-A peptide
-60 -40 -20 20 40 60
-50
-40
-30
-20
-10
0
10
(pA
/pF
)
V (mV)
control 1 (n=9)
AID peptide
0
-10
-20
-30
-40
-50
-60
-70
-80
pA
/pF
at 0 m
V
AID peptide (n=9)
AID W-A peptide (n=9)
0
-10
-20
-30
-40
-50
-60
-70
-80
pA
/pF
at 0 m
V
control group 1 (n=9)
AID peptide
0
-10
-20
-30
-40
-50
-60
-70
-80
pA
/pF
at 0 m
V
control group 2 (n=9)
AID W-A peptide
Aii Ai Aiii
Bii Bi Biii
CaV2.2 W391A peptide has INCREASED inhibitory effects
0.05
0.10
0.15
0.20
0.25
0.30
0.35
E(3
40)/
E(3
80)
control (n = 7)
AID peptide
AID W-A peptide
-10 mV 0 mV +10 mV
0
10
20
30
40
50
60
So
mato
sta
tin
% in
hib
itio
n
control (n = 7)
AID peptide (n = 7)
beta mutated AID (n = 7)
-10 mV 0 mV +10 mV
21
30 μm
AID peptide (n = 7)
SOM
SOM
control (n = 7)
AID W-A peptide (n = 7)
SOM
A
B
C
D
E
CaV2.2 W391A peptide has INCREASED inhibitory effects
Collaborator: Prof Cesare Usai, IIT Genova
22
Summary: CaV2.2 peptides are inhibitory agents
• Synthetic CaV2.2 peptides inhibit synaptic transmission and G protein
modulation
• CaV2.2 NT R52A,R54A peptide lacked inhibitory action: these data
may implicate arginines 52 and 54 as determinants for NT-I-II loop
interaction
• CaV2.2 AID W391A peptide had increased inhibitory action: bulky
tryptophan replaced by smaller alanine residue gives improved access?
• These data provide rational for designing improved inhibitory agents
Acknowledgements
Giovanna Bucci
Christian Vogl
Sumiko Mochida
23
Reading School of Pharmacy
top related