Introduction
Physiological and Pharmacological Characterisation
of a Novel Sarcolemmal Anionic Background
Conductance (IAB) in Heart
BY
John Joseph Borg PhD (Bristol)
•Cardiac arrhythmias are a major clinical problem for which traditional
pharmacotherapeutic approaches have targeted directly or indirectly
sarcolemmal cation conductances.
•The Cardiac Arrhythmia Suppression Trial (CAST) in 1991 (a placebocontrolled double-blind clinical trial of post-myocardial infarction (MI)
patients with asymptomatic ventricular arrhythmias), showed that sodium
channel blockers (encainide or flecainide) led to increased mortality in
patients.
•The SWORD trial (Survival with oral d-sotalol in patients with left
ventricular dysfunction after myocardial infarction) - increased mortality
Introduction
Introduction - Sarcolemmal Cl- channels are in a unique
electrophysiological situation
•Identification of new targets for drug action and tailoring of molecules to
fit these targets is highly desirable.
•A potential alternative to pharmacological inhibition of sarcolemmal
cation currents is to develop agents that target cardiac anionic channels.
•The equilibrium potential for Cl- (ECl)
in cardiac cells is about –50mV.
•Activation of cardiac ICl results in an
outwardly rectifying current which can:
•1) Shortens APD.
•2) Depolarise the RMP
•Three main cardiac Cl- currents
identified IClvol, IClcAMP, IClCa
Ca2+-activated chloride channels
Ca2+-activated chloride channels
Family
Types/sub-types
NH2
COOH
Structure
0utside
D1
Inside
CaCC
Ca2+-activatedchannels four
different families identified to date
D2
D3
D4
Expression
2 to 4 transmembrane
domains, still controversial
Many tissues including smooth muscle,
epithelium and heart
Conductance
1–10 pS
Permeation
I->Br->Cl-
Physiological mediators
Activation through elevation of [Ca2+]I
by neurotransmitters eg
noradrenaline, ATP & endothelin
1
ClC choride channels
ClC - voltage gated chloride channel
family
Family
ClC
Types/sub-types
Outside
L
C
B
I
H
D
E
F
M
N
Q
R
A
NH2
COOH
CBS 2
Each CLC subunit has 17 intramembrane
α helices labelled A to R where helix A is
cytoplasmic
P
K
G
Structure
O
J
Expression
Virtually ubiquitous
CBS 1
Conductance
Physiological mediators
CFTR (cystic fibrosis transmembane
regulator)
Family
Outside
CFTR
Structure
12 transmembrane domains
Expression
Epithelium, heart
NBD2
COOH
Conductance
Physiological mediators
Introduction - Sarcolemmal Cl- channels are in a unique
•The equilibrium potential for Cl- (ECl)
in cardiac cells is about –50mV.
•Activation of cardiac ICl results in an
outwardly rectifying current which can:
•1) Shortens APD.
•2) Depolarise the RMP
•Three main cardiac Cl- currents
identified IClvol, IClcAMP, IClCa
5–8 pS
Br->Cl->I-
Permeation
electrophysiological situation
Only one known several splice
variants
D D D
10 11 12
R
NBD1
NH2
Cell swelling, intracellular pH,
depolarisation, hyperpolarisation,
intracellular Ca2+
CFTR - Cystic fibrosis transmembrane
regulator
Types/sub-types
D7 D8 D
9
1–9 pS
I->Br->Cl-
Permeation
D1 D2 D3 D4 D5 D6
ClC1-7, Ka & Kb
in total nine members identified in
mammals
Phosphorylation by PKA, ATP
hydrolysis at Regulatory domain
Hypothesis
Therefore, activation of cardiac Cl- currents may affect
action potential duration (APD), and in disease states
could contribute to pathological changes in cardiac cell
function.
These observations have led to the formulation of the
following hypothesis.
Inhibition of Cl- conductances may lead to a prolongation
of repolarisation and an increase in the length of the
refractory period: effects which should provide inhibitors
of chloride channels with an anti-arrhythmic profile
similar to that of Class III anti-arrhythmic agents.
2
Introduction - IAB a novel background conductance in the heart
Introduction - Aims
Recently, a novel anionic background conductance termed -IAB- was
identified in rat ventricular myocytes and suggested to play a role in
APD (Spencer et al., 2000).
•To study the physiological relevance of this current by characterising IAB
in a species with a ventricular AP closer in profile to that of the human (ie
the guinea pig).
Thus the modulation of IAB could have potential clinical relevance.
•To investigate that IAB is pharmacologically distinct from other cardiac
chloride conductances.
In essence:
Inhibition of repolarising current - a Class III anti-arrhythmic approach.
•To determine the contribution of IAB in regulating the cardiac AP.
Increasing the magnitude of a repolarising current shortens the cardiac
AP -might lead to re-entry of arrhythmias
The patch-clamp recording technique
•To investigate whether the modulation of IAB affects the cardiac AP.
•To investigate whether chronic disease states affect IAB.
Whole cell recording
patch-electrode
cell-attached patch
cell
whole-cell
negative pressure
lift
lift
inside-out patch
outside-out patch
Methods - Electrophysiology (Whole Cell)
IAB
ICl,vol
Methods - Electrophysiology (whole Cell) - APs were
recorded at 35 °C
ICl,cAMP
Electrode AgCl
Bath Electrode
Na+, Ca2+ and
K+-free, Cs+,
[Cl]i was 21
mM
Na+, Ca2+ and
K+-free, Cs+,
[Cl]i was 21
mM
Na+, Ca2+ and
K+-free, Cs+,
[Cl]i was 21
mM
K+, Na+, Ca2+,
K2ATP,
glucose.
[Cl]i was 123
mM
Glass pipette
Myocyte
Na+, Ca2+and K+-free
NMDG-Cl-, Aspartate,
NO3- or ICs+, TEA+, Ba2+, and Cd2+
[Cl]o was 150 mM , [NO3-]o
, [Aspartate]0 & [I-]o was
135 mM.
Ca2+and K+-free.
Hyposmotic
external solution
prepared by
omitting 140 mM
sucrose. Final
osmolarity was
145 ± 5 mOsm,
[Cl]o was 78 mM.
ICl,cAMP – K+-free.
ICl,cAMP activated
with Forskolin (1
μM), [Cl]o was
153 mM.
Normal Tyrode’s, NaCl, KCl, CaCl2, MgCl2. Isotonic.
For anion substitution experiments NaCl was
replaced with NaAspartate or NaNO3-. [Cl]o was 155
mM , [NO3-]o & [Aspartate]0 & [I-]o was 134 mM.
3
Results - IAB - biophysical properties (guinea pig VM)
+80 mV
A
B
C
-80 mV
Cl- (b)
ASP (a)
500 ms
NO3- (c)
On the distinct nature of IAB
1 pA/pF
500 ms
D
NO3- (c-a)
E
Cl-
(b-a)
1 pA/pF
500 ms
Results - Further biophysical Characterisation of IAB in
guinea pig VM
(A)
1
Inward rectification of IClC-2 at -ve potentials
Im (pA/pF)
-90 mV
Time-dependent activation of
IClC-4 at +ve potentials
Time-dependent inactivation of
ICl,vol at +ve potentials
(B)
+ 70 mV
- 50 mV
- 50 mV
NO3-
NO3- (n=4)
500 ms
Aspartate
Erev (mV)
Relative
Permeability
-36.38 ± 0.8
-42.22 ± 2.77
-51.7 ± 0.89
500 ms
0
-0.1
-70
-1
-80 mV
I- (n=4)
0.1
0
ClINO3-
+80 mV
Cl- (n=5)
0.2
Im (pA/pF)
Differences between IAB and other Cl- currents
• instantaneous-activation of current (unlike IClC,4)
• no time-dependent inactivation at +ve potenntials (unlike Icl,cvol)
• Outwardly rectifying (IClC,2 is inwardly rectifying)
• Carried by different anions (N03- is a better charge carrier than Cl-; this differentiates IAB
from the voltage-gated CLC-4 channels)
-50
Vm (mV)
-30
-10
Using the modified GHK eqn: NO3->I->Cl-
1.57 ± 0.17
2.05 ± 0.19
Permeability sequence of IAB is different from that of ICl,cAMP (Br->Cl->I->F-); ICl,vol (SCN->I->NO3->Br>Cl->F-) and ICl,Ca (SCN->I->Br->Cl-)
Representative IAB current traces elicited by depolarising voltage-ramps
Results - IAB (NO3- - sensitive currents recorded from guinea pig
VM, rat atrial and VM)
+ 70 mV
A
B
-90 mV
0.6
500 ms
0.4
0
ASP (a)
pA/pF
pA/pF
NO3- (b)
Nitrate-sensitive difference current
0.2
NO3- (b-a)
pA/pF
1
1.0
- 50 mV
- 50 mV
No significant difference was observed
between groups (P>0.05; ANOVA)
0.5
0
-1
-75 -50 -25 0 25 50 75
Vm (mV)
0.0 Guinea pig VM Rat VM
(n=9)
(n=4)
Rat AM
(n=5)
These results suggest that IAB night be homogeneously distributed in heart. This is not the case with
ICl,vol and ICl,cAMP
4
Results - IAB (Effect of hypertonic conditions, PKA and PKC
inhibition and ~20 nM intracellular Ca2+ on IAB in guinea pig VM )
+ 70 mV
- 50 mV
-90 mV
1
Ca2+and
pA/pF
NO3- (b)
Na+,
K+-free
NMDG-NO3-,
Aspartate.
Hypertonicity ↑ with
63 mM Sucrose
0
B
1.0
- 50 mV
0.6
500 ms
ASP (a)
0.4
0.2
NO3- (b-a)
0.0
-1
pA/pF
A
pA/pF
Na+, Ca2+, K+,
Mg-ATP, trisGTP -free, 1
mM Cs EGTA,
H7-DHC (6
μM)
Results - IAB (Effect of hypertonic conditions, PKA and PKC
inhibition and calcium chelation on IAB in guinea pig VM )
-75-50-25 0 25 50 75
Vm (mV)
0.0
These results suggest that IAB is not ICl,vol, ICl,cAMP and ICl,Ca
Results - I
AB
0.5
(Effect of GTP-y-S (intracellular) on IAB in guinea pig VM )
CTRL (n=9)
intracellular H7-DHC
hypertonic conditions (n=4)
Results - IAB’s Pharmacology is distinct from other Cl- currents in
the heart
+ 70 mV
1
These results suggest that IAB is not
sensitive to activation by g-proteins
which differentiates IAB from ICl,vol,
and ICl,cAMP
500 ms
pA/pF
0.4
ASP (a)
0.2
NO3- (b-a)
B
0.6
DIDS 50 μM
0.4
0.2
NO3-
TAMOX 100 μM
0.4
0.2
NO3-
0.0
0.0
-0.2 -75 -50 -25 0 25 50 75
Vm (mV)
-0.2 -75 -50 -25 0 25 50 75
Vm (mV)
0.0
-1
For A and B similar results were observed in
five myocytes
• DIDS reported to inhibit ICl,vol, ICl,Ca
•Tamoxifen reported to inhibit ICl,vol
-75 -50 -25 0 25 50 75
(mV)
C
pA/pF
0
CTRL exp showing that GTP-y-S
potentates ICa,L in guinea pig VM, and
therefore GTP-y-S is active under
these experimental conditions
-2
-4
-6
0 mV
500 ms
-40 mV
Results - IAB’s Pharmacology is distinct from other Cl- currents in
the heart
0.6
pA/pF
pA/pF
NO3- (b)
0
A
0.6
pA/pF
-90 mV
B
- 50 mV
- 50 mV
pA/pF
A
Results - IAB’s Pharmacology is distinct from other Cl- currents in
the heart - The search for Novel modulators !
Type I pyrethroids
CTRL
Test agent
O
O
O
O
F3C
Cl
O
0.4
Bioallethrin
0.2
0.0
O
F3
C Cl
Gd3+ (1 mM)
(n=5)
Picrotoxin (1 mM)
(n=5)
Chlorotoxin (1 µM)
(n=4)
• Gd3+ reported to inhibit the voltage-gated
chloride channels CLC-0 & CLC-1, ICl,vol, the nonselective cation channel (INSC), the sodiumcalcium exchanger current (INCX]) and the
stretch-activated cation current (ISAC)
• Picrotioxin reported to inhibit ligand-gated Clcurrents (GABAA)
•Chlorotoxin reported to small conductance Clchannels
F
O
F
Bifenthrin
O
O
O N
F
O
F
Tefluthrin
Tetramethrin
Type II pyrethroids
Cl
Cl
O CN
O
F
O
Cyfluthrin
Cl
Cl
O CN
O
O CN
O
O
O
Cypermethrin
Fenpropathrin
Pyrethroids have been shown to decrease the rate of taurine efflux in C6 and HELA cells
Disparate chemical pharmacophores (stereo chemistry)
5
Results - Effects of Pyrethroids on IAB
B
+ 70 mV
- 50 mV
- 50 mV
-90 mV
1.0
0.8
CYP (c)
NO3- (b)
ASP (a)
0
0.8
0.4
0.2
NO3- (b-a)
0.0
-1
-75
500 ms
Anomalous
mole
fraction
effect
1.2
NO3
ASP
-
-50
-25
0
25
Vm (mV)
50
FENP
CYP
TEF
10 µM CYP
EC50 (µM)
nH
3.01 ± 0.1
7.02 ± 0.2
4.0 ± 0.3
1.0 ± 0.3
0.9 ± 0.3
2.1 ± 0.9
0.6
All three compounds maximally activated
IAB to the same extent (P>0.05; ANOVA)
75
0.4
ASP
1.0
pA/pF
(FENP)
(CYP)
(TEF)
1.0
CYP (c-a)
0.6
pA/pF
pA/pF
1
pA/pF
A
Results - Effects of Pyrethroids on IAB
+60 mV
0.8
0.2
0.01
0.1
1
10
Concentration (μM)
100
0.6
n>3 for each data point
0.4
0
200
400
600
800
Time (s)
1000
1200
1400
Results - Effects of 300 µM TET on IAB
Results - Effects of Pyrethroids on anion insensitive residual current
This exp is a ctrl showing that CYP, FENP
and TEF do not activate an anion
insensitive residual current or a leak
current.
0.4
Increase in Im (pA/pF)
pA/pF
0.6
0.2
0.0
CTRL
300 µM TET
(n=5)
0.10
No significant difference was observed in
the absence and presence of pyrethroid
agents (P>0.05; Student’s t-test)
0.05
0.0
This exp shows that not all pyrtheroids
effect IAB. Therefore differences in the
structures between pyrethroids might
hint to a Pharmacophore.
CYP
(n=5)
FENP
(n=6)
TEF
(n=5)
Results - Effects of 100 µM FENP, CYP and TET on ICl,vol
(A)
-60 mV
2
HYPO + FENP
ISO
5s
-60
-40
-20
-2
20 40
Vm (mV)
**
4
2
60
0
(B)
(n=5)
(C)
pA/pF
Note: in Guinea pig VM only ICl,vol and
ICl,cAMP have been recorded.
HYPO
4
HYPO + CYP
HYPO
4
pA/pF
-50 mV
pA/pF
+60 mV
pA/pF
Are the effects of pyrtheroids on IAB
selective? Or do they have other effects on
other Cl- currents (ICl,vol and ICl,cAMP) ?
ISO
HYPO
HYPO + FENP
2
HYPO + TET
HYPO
4
2
ISO
-60
-40
-20
20
-2
40
Vm (mV)
60
ISO
-60
-40 -20
20
-2
40
60
Vm (mV)
6
Results - Effects of FENP and DIDS on ICl,vol
Results - Effects of 100 µM FENP, CYP, TET, TEF on ICl,cAMP
CTRL
FSK
FSK + CYP
CTRL + CYP
(B)
Isotonic
Hypotonic
Hypotonic & DIDS 50 µM
1
(n=4)
10
Concentration (µM)
2
CTRL + CYP
CTRL
3
-60
2
20
40
Vm (mV)
*
4
2
60
0
1
(n =5)
(n=5)
FENP inhibits ICl,vol with an IC50 of 26.2 ± 0.3 µM
and a Hill coefficient of 2.1 ± 0.1
-20
-2
0
100
-40
B) shows a CTRL exp where ICl,vol is inhibited
by DIDS. This shows that under these
conditions ICl,vol has been activated and not a
hypotonically induced cation current.
B
pA/pF
(n=4)
0
FSK + CYP
FSK
4
-60
-40
-20
4
FSK
2
CTRL II
CTRL I
-2
20
40
pA/pF
(n=5)
50
(pA/pF
% Inhibition
(n=3)
pA/pF
(n=5)
100
pA/pF
A
(A)
CTRL
FSK
CTRL
4
2
60
0
Vm (mV)
(n =4)
B is a CTRL exp showing that upon removing FSK from perfusate ICl,cAMp reverses back to CTRL levels
Results - Effects of 100 µM FENP, CYP, TEF & TET on
background currents after FSK was removed from the bath solution
Results - Effects of glibenclamide on ICl,cAMP
Control (n=5)
Forskolin 1 µM (n=5)
Forskolin 1 µM + Glibenclamide 100 µM (n=5)
100
shows a CTRL exp where ICl,cAMP is inhibited
by GLB. This shows that under these conditions
IClcAMP has been activated
3.0
60
Im (pA/pF)
% Increase
80
40
20
0
CYP
(n=5)
FENP
(n=5)
TET
(n=4)
TEF
(n=4)
2.0
1.5
1.0
0.5
Although comparisons of this exp with the effects of pyrethroids on nitrate
sensitive currents (IAB) cannot be made (due to the different recording conditions
used to isolate the currents), a common pattern of pyrethroid activity on basal
currents can be observed
Results - Effect of anion substitution on APs
2.5
0.0
Results - Effect of 10 µM TEF on APs
A
100
155 mM [Cl-]0
134 mM [NO3-]0
Anion substitution exps identical to voltageclamp exps show that IAB can shorten APD
B
155 mM [Cl-]0
155 mM [Cl-]0 + TEF
134 mM [ASP]0
134 mM [ASP]0 + TEF
Vm (mV)
50
0
-50
-100
250 ms
APD90 by ~21%
250 ms
APD90 by ~21%
250 ms
APD90 by ~52%
A shows that TEF shown to increase IAB (therfore increasing an outwardlyrectifying current) shorten APD.
B shows that when IAB is inhibited (by replacing Cl- with ASP) TEF does not
shorten APD. - lengthening of APD is due to TEF’s reported effect of slowing INa
inactivation
7
Results - Incorporation of IAB into a guinea pig ventricular cell
model
Reports have shown that anion substitution
and pyrethhroid agents may affect
sarcolemmal cationic conductances
100
Control
IAB incorporated
0.7
50
•ASP has been shown to inhibit ICa,L
0.6
experimental
0.5
simulation
•Some pyrethroids shown to modulate
cardiac INa and ICa,L & ICa,T
pA/pF
Vm (mV)
0.4
0
0.2
0.1
-50
•Therefore to overcome the limitation that
the interventions used during AP Exps
were not entirely selective for IAB, AP
simulations were conducted
0.3
0.0
-0.1
-100
250 ms
-80 -40 0
40
Vm (mV)
80
APD90 by 12 % (34 ms)
This result is important because it shows that a background Cl- conductance can modulate APD
Results - Incorporation of IAB into a guinea pig ventricular cell
model
•Incorporation of IAB in the ventricular AP model suggests that IAB plays a
role in APD.
•IAB affected APD by a similar magnitude therapeutically used Class III
anti-arrhythmic agents sotalol and amiodarone at clinically relevant
serum concentrations.
Results obtained show that Increasing IAB can reduce
APD
• Shortening of APD shown to precipitate arrhythmias VT, VF, short QT syndrome
•Sotalol increases APD90 by 20 to 40 ms (Groh WJ et al., 1995),
•Amiodarone increases APD90 by 12 to 15 ms in rabbits (Kirchhof P et
al., 2003)
•1st time that a background Cl- current has been incorporated into a
guinea pig ventricular AP model.
•Routine incorporation of IAB in ventricular AP models
Results - Pyrethroids are pro-arrhythmic
1.6
FOC (arb)
•Diabetes and Hypertension have been show to induce
cardiac remodeling of ion channels
TEF
C
1.4
B
CTRL
1.8
% CV
PCCF (arb)
A
Results - Does disease affect IAB
1.5
CTRL
Test Agent
*
*
*
1.0
0.5
0
400
Time (s)
CTRL
800
1200
0.0
FENP
CYP
TEF
TET
TEF
• Decreased expression of K+ channels have been
suggested to result in the morphological changes in the
AP
As suggested by AP studies pyrethroids
are pro-arrhythmic
1.6
1.3
1 (s)
8
Results - Does disease affect IAB
0.6
Summary
The present study has shown that:
(CTRL)
(Disease)
pA/pF
•IAB was found to exist in guinea pigs.
0.4
0.2
0.0
Obesity and Diabetes
Hypertension
This exp shows that
unlike other K+ channels
the current density of IAB
is not affected by
diabetes and
hypertension
•This is important as guinea pig ventricular myocyte electrophysiology is
similar to that of the human, and therefore IAB could be present in
humans. Thus being a potentially new pharmacological target in the
heart.
•IAB was found to be a time-independent, outwardly rectifying current that
is highly permeable to NO3-. IAB has a relative permeability sequence of
NO3->I->Cl->aspartate.
Summary
•IAB was not blocked by DIDS, TAMOX, Gd3+, Picrotoxin and chlorotoxin,
or hypertonicity, or by the non-selective protein kinase inhibitor H7-DHC
6 μM (inhibits both PKA and PKC).
•IAB has a distinct pharmacology from the currently identified Clconductances in the heart.
•Collectively the results suggest that the molecular identity underlying IAB
is distinct to other Cl- currents in the heart.
•Activators of IAB indicate its potential value as a novel pharmacological
target in the heart:
•Inhibition of IAB could be of value in producing a modest Class III
antiarrhythmic effect.
9