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Kv7 CHANNELS IN Kv7 CHANNELS IN SMOOTH MUSCLE SMOOTH MUSCLE AS THERAPEUTIC AS THERAPEUTIC
TARGETS FOR TARGETS FOR VASCULAR AND VASCULAR AND
AIRWAY DISEASESAIRWAY DISEASES
Kenneth L. Byron, Ph.DKenneth L. Byron, Ph.DProfessor of PharmacologyProfessor of PharmacologyLoyola University ChicagoLoyola University Chicago
From: P.R. Wheater, H.G. Burkitt, V.G. Daniels, Functional Histology , 1979
DISCLOSURE
• U.S. Patents issued:– Patent 8686017 “Methods of using
proteinacious channels to identify pharmaceutical treatments and risks, and treatments resulting therefrom”
– Patent 20,140,155,368 “Combination pharmaceuticals and methods thereof using proteinacious channels as treatments for medical conditions”
Vascular Smooth MuscleVascular Smooth MuscleV1a
KidneyV2AVPAVP
AVPAVP
PituitaryAVPAVP
↓ Blood Pressure
↑ PlasmaOsmolality
Arginine-Vasopressin (AVP): A pituitary hormone that regulates water balance and blood pressure
Plasma AVP Concentrations
00--500 pM500 pM
Change in OsmolarityChange in Osmolarity Change in Blood PressureChange in Blood Pressure
00--12 pM12 pM
Baylis PH, Clin.Endocrinol.Metabol.1983
00 500pM500pM
Physiological rangePhysiological range
10101010--1212
[AVP] (M)[AVP] (M)
00100100200200300300400400500500600600700700800800
101010101010101010101010--55--1111 --1010 --99 --88 --77 --66
Peak CaPeak Ca
2+2+ii (nM
)(nM
)
ECEC5050=5nM=5nMECEC5050=150pM=150pM
AVP Concentration-dependent Ca2+ Signaling in Vascular Smooth Muscle Cells (A7r5 cells)
00
55
1010
1515
2020Sp
ikin
g Fr
eque
ncy
(sSp
ikin
g Fr
eque
ncy
(s-- 11))
Action potentials
Novel AVP signaling pathway identified in A7r5 rat aortic smooth muscle cell line
AVP
V1aR
DAG + IP3PIP2
Release of SR Ca2+
Store
PLC
High (EC50=5 nM)AVP Pathway
Ca2+ Spikes
PLD
PKC
CaL
K+
Low (EC50=150pM) AVP Pathway
∆EM Ca2+
_ K v
Isolated Kv currents are non-inactivating.
Brueggemann et al. Am J Physiol Heart Circ Physiol 292:1352-1363, 2007
Dr. Lioubov Brueggemann
Isolated Kv currents activate at very negative voltages and are suppressed by AVP
V0.5 = -38.0 ± 1.6 mVs = 8.3 ± 0.4 mVn=21
Brueggemann et al. Am J Physiol Heart Circ Physiol 292:1352-1363, 2007
Control
AVP
AVP-sensitive Kv currents in A7r5 cells have characteristics of neuronal “M currents”
1. Non-inactivating delayed rectifier K+ currents that activate over a relatively negative voltage range.
2. Inhibited in response to activation of G protein-coupled receptors, resulting in increased electrical excitability.
Are the vascular smooth muscle currents mediated by KCNQ (Kv7) channels?
Kv currents are inhibited by selective KCNQ channel blockers linopirdine & XE991
and reversibly activated by selective KCNQ channel activator flupirtine
Brueggemann et al. Am J Physiol Heart Circ Physiol 292:1352-1363, 2007
KCNQ5 is expressed in A7r5 cells.
Brueggemann et al. Am J Physiol Heart Circ Physiol 292:1352-1363, 2007
Knocking down KCNQ5 expression or function abolishes the AVP-
sensitive KCNQ currents.
V, mV
I, pA/pF
-80 -40 0 40
0.6
control, n=6 Q5shRNA, n=8
V, mV-80 -40 0 40
I, pA/pF
0.0
0.2
control, n=13 dnQ5, n=18
Action potentials
Novel AVP signaling pathway identified in A7r5 rat aortic smooth muscle cell line
AVP
V1aR
DAG + IP3PIP2
Release of SR Ca2+
Store
PLC
High (EC50=5 nM)AVP Pathway
Ca2+ Spikes
PLD
PKC
CaL
K+
Low (EC50=150pM) AVP Pathway
∆EM Ca2+
_ KCNQ
5KC
NQ5
Vasoconstriction?
Outer diameters ~ 300-400 µm
Vasoconstrictor responses measured in pressurized rat mesenteric arteries
AVP concentration-
dependent constriction of rat mesenteric
arteries
30 pM AVP
10 nM AVP
Henderson & Byron, J Appl Physiol 102:1402-1409, 2007
T im e ( s e c o n d s )3 0 0 0 3 5 0 0 4 0 0 0 4 5 0 0 5 0 0 0 5 5 0 0 6 0 0 0 6 5 0 0 7 0 0 0 7 5 0 0
30pM AVP 10nM AVP 60mM KCl
0
Vess
el O
uter
Dia
met
er (u
m)
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
a
b
c
AVP
V1aR
DAG + IP3PIP2
Release of SR Ca2+
Store
PLC
10 nMAVP Pathway
Ca2+ Influx
PLD
PKC
CaL
K+
∆EMCa2+
_
KCNQ
KCNQ
Vasoconstriction
30 pMAVP Pathway
Novel AVP signaling pathway
AVP
V1aR
DAG + IP3PIP2
Release of SR Ca2+
Store
PLC
10 nMAVP Pathway
Ca2+ Influx
PLD
PKC
CaL
K+
30 pM AVP Pathway
∆EM Ca2+
_ KCNQ
KCNQ
Vasoconstriction
M-currents measured in freshly isolated mesenteric artery myocytes
Kv currents are enhanced by KCNQ channel activators (retigabine and flupirtine) and abolished by KCNQ blockers(XE991 and linopirdine).
2 sec
40 p
Acontrol 10 M flupirtine 10 M XE-99116 mV
-84 mV
-4 mV -4 mV
Control
10 M Flupirtine
V, mV-80 -60 -40 -20 0 20
I, pA/pF
0.4
10M XE-991
Mackie et al., JPET 2008
AVP suppresses KCNQ currents, but not4-AP-sensitive Kv currents
ControlControl100 pM AVP100 pM AVP100 pM AVP + 10 100 pM AVP + 10 μμM FlupirtineM Flupirtine
100 pM AVP + 10 100 pM AVP + 10 μμM LinopirdineM Linopirdine
0.40.4
--2020--4040--6060--8080V (mV)V (mV)
I (pA/pF)I (pA/pF)
n = 4 cellsn = 4 cells
V (mV)V (mV)--8080 --6060 --4040 --2020 00 2020 4040
1010
2020
I (pA/pF)I (pA/pF)
ControlControl100 pM AVP100 pM AVP2 mM 42 mM 4--APAP
n = 3n = 3--6 cells6 cells
G/Gmax
V, mV-80 -60 -40 -20 0 20 40-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
KCNQV0.5 = -34.3 mVs = 6.5 mVn = 27
Kv_4-APV0.5 = +5.1 mVs = 8.2 mVn=8
Boltzmann fits
Mackie et al., JPET 2008
KCNQ channel activator flupirtine: concentration-dependent vasodilation
0 10 20 30 40% o
f Max
imal
Dila
tion
020406080
100
Flupirtine (μM)Out
er V
esse
l Dia
met
er (u
m)
200
220
240
260
280
300
320
340
10 min
10 μM
20 μM
30 μM
40 μM flupirtine
0 μM
30pM AVP
Mackie et al. Mackie et al. JPETJPET 20082008
In Vivo Effects of KCNQ Channel Modulators
Linopirdine (n=3)( )Linopirdine (n=3)( )
60608080
100100120120
Flupirtine (n=3)( )Flupirtine (n=3)( )
% of % of Basal Basal MAPMAP
***
**
60608080
100100120120
% of % of Basal Basal MVRMVR
* *
* *
*
000.0
10.0
10.0
30.0
30.1
00.1
00.3
00.3
01.0
01.0
03.0
03.0
080809090
100100110110120120
% of % of Basal Basal
HRHR **
Dose (mg/kg i.v)Dose (mg/kg i.v) Mackie et al. Mackie et al. JPETJPET 20082008
Celecoxib(Celebrex®)
Rofecoxib(Vioxx®)
COX-2 Inhibitors
Celebrex®, but not Vioxx®, activates native or overexpressed vascular KCNQ channels
15105Time, min
0
510152025303540
10M celecoxib
I at -2
0 m
V, p
A/p
F
A7r5 cell expressing hKCNQ5
V, mV-80 -40 0 40
I, pA/pF
0.5
1.0
1.5
control10M rofecoxib10M celecoxib
A7r5 cell
Out
er D
iam
eter
(mic
rons
)
150
200
250
300
350
400
450
RofecoxibCelecoxib
100pM Vasopressin
10 min
0
20
40
60
80
100
120
Treatment
% o
f Dila
tion
20uM Rofecoxib 20uM Celecoxib
*
t-test, P=<0.001
Celebrex® but not Vioxx® can relax rat mesenteric arteries pre-constricted with AVP
Brueggemann et al. Brueggemann et al. Mol Pharm Mol Pharm 20092009
KCNQ SubtypeKCNQ Subtype
Rat BrainRat Brain
MASMCMASMC
500 500 --
500 500 --
11 22 33 44 55RTRT--PCRPCR
KCNQ channel expression in mesenteric artery myocytes: KCNQ1, KCNQ4, & KCNQ5 are detected by RT-PCR
Mackie et al., JPET 2008
hKCNQ4
-2
0
2
4
I, nA
2 s
hKCNQ5
2 s-2
0
2
4
I, nAhKCNQ4/5
2 s-2
0
2
4
I, nA
A
B
100 M diclofenac
10864200
1
2
3
time, min
I, nA
hKCNQ4
hKCNQ4/5
hKCNQ5
C
D
V, mV-100 -80 -60 -40 -20 0
G/G
max
0.0
0.2
0.4
0.6
0.8
1.0
hKCNQ4/5hKCNQ5hKCNQ4
I/Ico
ntro
l
0.0
0.5
1.0
1.5
2.0
[diclofenac], M10 100
hKCNQ4/5hKCNQ5hKCNQ4
Diclofenac distinguishes between overexpressed homomeric and heteromeric KCNQ4 & KCNQ5 channels
Brueggemann et al. Mol. Pharmacol., 79: 10-23, 2011.
A7r5 cells
MesentericArtery myocytes
Diclofenac distinguishes between natively expressed homomeric and heteromeric KCNQ4 & KCNQ5 channels
Brueggemann et al. Mol. Pharmacol., 79: 10-23, 2011.
Patent number Company
Target channel Chemical class Representative compound
6,326,385 Icagen KCNQ2/Q3 N-aryl benzamide
ICA-27243 analogs
6,372,767 Icagen KCNQ2 Benzanilides and 2-substituted-5-aminopyridines
6,495,550 Icagen KCNQ2 Pyridine-substituted benzanilides
6,989,398 Icagen KCNQ2 Benzanilides
6,605,725 Icagen KCNQ2 Benzanilides
6,737,422 Icagen KCNQ2 Benzanilides
6,593,349 Icagen KCNQ2 Bisarylamines
7,741,332 Icagen KCNQ2 Fused ring heterocycles
7,223,768 Icagen KCNQ2 Fused ring heterocycles
6,469,042 BMS KCNQ2, KCNQ2/Q3 Fluoro oxindole derivatives
BMS-204352 analogs
6,855,829 BMS KCNQ2, KCNQ5
3-fluoro-2-oxindole and 2,4-disubstituted pyrimidine-5-carboxamide
7,632,866 Tel Aviv U. KCNQ2/3, Q1, Q1/E1
Derivatives of N-phenylanthranilic acid and 2-benzimidazolone as potassium channel and/or neuron activity modulators
LIST OF US PATENTS ISSUED PRIOR TO 2011 THAT CLAIM COMPOUNDS ACTIVATING KCNQ CHANNELS
ML213
N-mesitylbicyclo[2.2.1]heptane-2-carboxamide
A small molecule activator of KCNQ2 and KCNQ4 channelsHaibo Yu, Meng Wu, Corey Hopkins, Julie Engers, Steve Townsend, Craig Lindsley, Owen B McManus, and Min Li.
“ML213 was identified following a high throughput fluorescent screen of the Molecular Libraries Small Molecule Repository (MLSMR) library and structure activity relationship (SAR) studies using fluorescent and electrophysiological assays to determine potency and selectivity of test compounds. ML213 is a potent activator of potassium voltage-gated channel, KQT-like subfamily, member 2 (KCNQ2) (Kv7.2, EC50 = 230 nM) and KCNQ4 (Kv7.4, EC50 = 510 nM) and selective against the other members of the KCNQ family of ion channels (KCNQ1, KCNQ3 and KCNQ5).”
Probe Reports from the NIH Molecular Libraries Program
54321Time (s)
-1.0
-0.5
0
0.5
1.0
I, nA control
54321Time (s)
-1.0
-0.5
0
0.5
1.0
I, nA 10 µM ML213hKv7.4/7.5
hKv7.4controlI, nA
54321Time (s)
-3-2-1012345
0
10 µM ML213I, nA
54321Time (s)
-3-2-1012345
0
hKv7.5
54321Time (s)
-3-2-1012345I, nA control
54321Time (s)
-3-2-1012345I, nA 10 µM ML213
ML213
leftw
ard
shift
of a
ctiv
atio
n cu
rve
(∆V
0.5)
, mV
[ML213], µM0.1 1 10
0
10
20
30
40
50hKv7.4 EC50= 1.50.1µMhKv7.5 EC50= 3.40.4µMhKv7.4/7.5 EC50= 3.20.4µM
Cerebral Aneurysm and Subarachnoid Cerebral Aneurysm and Subarachnoid hemorrhage (SAH).hemorrhage (SAH).
Hypothetical model of the role of KCNQ Hypothetical model of the role of KCNQ channels in cerebral vasospasm. channels in cerebral vasospasm.
Bharath Mani’s project
Ca
L
KC
NQ
Em Ca
L
KC
NQ
Vasospasm
Ca2+
Em
Spasmogen
R
PKC
StrokeStroke
K+KCNQ channel activators
1 2 3 4 5
KCNQ Subtype
500 bp
1 2 3 4 5
KCNQ Subtype
500 bp
KCNQ channels are expressed and KCNQ channels are expressed and functional in basilar artery myocytes.functional in basilar artery myocytes.
ControlFlupirtine 10µMXE991 10µM
V, mV-80 -60 -40 -20 0 20
I, pA/pF
0.2
0.3
*
***
* **
***
*
Celebrex® prevents or reverses vasoconstriction in basilar arteries
VehicleC
Vess
el o
uter
dia
met
er (µ
m)
200
250
300
350
Celecoxib 30µMSerotonin 75nM Serotonin 75nM
Wash outCLCXB
20 minVess
el o
uter
dia
met
er (µ
m)
200
250
300
350
Celecoxib 30µMSerotonin 75nM Serotonin 75nM
Wash outCLCXB
20 min
C
Celecoxib
1µM
10µM20µM
30µM
Serotonin 75nM
Vess
el o
uter
dia
met
er (µ
m)
275
300
325
350
375
400
10 min
[celecoxib] µM0 5 10 15 20 25 30
% re
laxa
tion
20
40
60
80
100
Serotonin 75nM AVP 100pM Endothelin 100pM
A B
No Surgery aCSFControl
SAHVehicle
SAHRetigabine
SAHCelecoxib
No Surgery aCSF Control
SAH Vehicle
SAH Retigabine
SAH Celecoxib
Vess
el O
uter
dia
met
er (µ
m)
0
50
100
150
200
250
** **
Retigabine or Celecoxib prevents basilar artery vasospasm in a rat model of SAH
Mani et al. (2013) J. Cardiovasc. Pharmacol. 61, 51-62
KCNQ CHANNELS IN VASCULAR SMOOTH KCNQ CHANNELS IN VASCULAR SMOOTH MUSCLE CELLS AS PHYSIOLOGICAL AND MUSCLE CELLS AS PHYSIOLOGICAL AND
THERAPEUTIC TARGETSTHERAPEUTIC TARGETS
AVP
V1aR
DAG + IP3PIP2
Release of SR Ca2+
Store
PLC
High (EC50=5 nM)AVP Pathway
Ca2+ Influx
PLD
PKC
CaL
K+
Low (EC50=150pM) AVP Pathway
∆EM Ca2+
_ KCNQ
5KC
NQ5
Vasoconstriction
KCNQ CHANNELS IN KCNQ CHANNELS IN AIRWAYAIRWAY SMOOTH SMOOTH MUSCLE CELLS AS PHYSIOLOGICAL AND MUSCLE CELLS AS PHYSIOLOGICAL AND
THERAPEUTIC TARGETS?THERAPEUTIC TARGETS?
Kv7 CURRENTS IN AIRWAY SMOOTH MUSCLE CELLS Kv7 CURRENTS IN AIRWAY SMOOTH MUSCLE CELLS ARE ENHANCED BY FLUPIRTINE AND CELECOXIB.ARE ENHANCED BY FLUPIRTINE AND CELECOXIB.
A B
DC
-64mV-16mV
-104mV
-50
0
50
100
2 s
I, pA
G/G
max
V, mV-80 -60 -40 -20 0
0.0
0.2
0.4
0.6
0.8
1.0control, n=11 10 µM flupirtine, n=7
control 10 µM flupirtine 10 µM XE-991
V, mV-80 -60 -40 -20 0
I, pA/pF
1
3
02468
101214 10 µM celecoxib
5 min
I, pA
SUPPRESSION OF Kv7 CURRENTS IN AIRWAY SUPPRESSION OF Kv7 CURRENTS IN AIRWAY SMOOTH MUSCLE CELLS BY METHACHOLINE AND SMOOTH MUSCLE CELLS BY METHACHOLINE AND
HISTAMINE IS REVERSED BY FLUPIRTINE.HISTAMINE IS REVERSED BY FLUPIRTINE.
V, mV-80 -60 -40 -20 0
I, pA/pF
0.4
0.6
0.8control, n=4 100nM MC, n=4 10M XE-991, n=3
control, n=3 30M His, n=3 10M XE-991, n=3
V, mV-80 -60 -40 -20 0
I/Ic
0.0
1.0
0
2
4
6
100 nM methacholine
10M F
2 min
I at -2
0mV,
pA
A B C
I/Ic at -20mV
0.0
0.5
1.0
C His His+ F XE-991 C MC MC+ F XE-991
* *
D
##
PRECISIONPRECISION--CUT LUNG SLICES: FUNCTIONAL CUT LUNG SLICES: FUNCTIONAL EFFECTS OF KCNQ CHANNEL MODULATION ON EFFECTS OF KCNQ CHANNEL MODULATION ON
AIRWAY DIAMETERAIRWAY DIAMETER
100μm 100μm 100μm 100μm 100μm
a b c d eA
10 μM retigabine
time, min0 20 40 60 80 100
area
μm
2
0
10000
20000
30000
40000
50000
60000
1 μM methacholine
a
b
c
d
e
B
CONCENTRATION-DEPENDENCE OF REIGABINE-INDUCED RELAXATION OF RAT AIRWAYS
Brueggemann et al. Am J Physiol Lung Cell Mol Physiol 2014
A B
time, min0 20 40 60 80 100 120 140
area
, µm
2
15000
20000
25000
30000
35000MC
retigabine
MCar
ea, µ
m2
time, min0 20 40 60 80 100 120 140
0
50000
100000
150000
200000
250000 MC MC
A B
time, min0 20 40 60 80 100 120 140
area
, µm
2
15000
20000
25000
30000
35000MC
retigabine
MC
time, min0 20 40 60 80 100 120 140
area
, µm
2
15000
20000
25000
30000
35000MCMC
retigabineretigabine
MCMCar
ea, µ
m2
time, min0 20 40 60 80 100 120 140
0
50000
100000
150000
200000
250000 MC MCar
ea, µ
m2
time, min0 20 40 60 80 100 120 140
0
50000
100000
150000
200000
250000 MCMC MCMC
C
230
nM M
C -in
duce
dco
nstri
ctio
n (r
elat
ive
to1st
MC
- indu
ced
cons
trict
ion)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
MC MC+R MC+DMSO MC+DMC MC+ZnPyr
* *
CC
*
KCNQ POTASSIUM CHANNEL ACTIVATORS ATTENUATE METHACHOLINE-INDUCED CONSTRICTION OF RAT AIRWAYS.
Brueggemann et al. Am J Physiol Lung Cell Mol Physiol 2014
PROFOUND CONSTRICTION OF HUMAN AIRWAYS PROFOUND CONSTRICTION OF HUMAN AIRWAYS INDUCED BY Kv7 CHANNEL BLOCKER XE991; INDUCED BY Kv7 CHANNEL BLOCKER XE991;
ATTENUATION OF HISTAMINEATTENUATION OF HISTAMINE--INDUCED AIRWAY INDUCED AIRWAY CONSTRICTION BY FLUPIRTINE.CONSTRICTION BY FLUPIRTINE.
control 50 nM Hist 50 nM Hist+100 μM F
washout 10 μM XE991
time, min0 10 20 30 40 50
area
, μm
2
0
500
1000
1500
Hist Hist+F 10 μM XE991
A
B
FORMOTEROLDrug class: Long-acting β2-adrenergic agonist (LABA)
Use: Bronchospasm, Exercise-induced asthma
Regular treatment with both long- and short-acting β2-agonists results in tolerance to their bronchoprotective effects. Twice daily administration of long-acting β2-agonists results in blunted responses to repeated doses of short-acting β2-agonists , as with rescue inhalers used in the setting of an acute asthma attack.
DESENSITIZATION TO LABAs
FORMOTEROL ALONE TRANSIENTLY RELAXES RAT AIRWAYS, WHEREAS IN COMBINATION WITH
RETIGABINE IT INDUCES GREATER AND MORE SUSTAINED BRONCHODILATION
Brueggemann et al. Am J Physiol Lung Cell Mol Physiol 2014
COMBINING RETIGABINE WITH FORMOTEROL IMPROVES RELAXATION OF ASTHMATIC
HUMAN AIRWAYSH = 25 pM histamineFF = 30 pM formoterol fumarateR = 10 µM retigabine
KCNQ (Kv7) K+ channels contribute to stabilization of resting membrane potential and are essential intermediates in vasoconstrictor and bronchoconstrictor signal transduction in vascular and airway smooth muscle, respectively.
In intact arteries, drugs that activate vascular KCNQ currents induce vasodilation. Drugs that inhibit KCNQ currents are vasoconstrictors. These effects contribute to changes in arterial blood flow and systemic blood pressure.
Drugs currently in clinical use may have unexpected cardiovascular side effects due to previously unrecognized effects on vascular KCNQ channels. To predict these cardiovascular side effects, screening of drugs for actions on vascular KCNQ currents will be required. Some drugs may distinguish among KCNQ channels formed from different subunit combinations.
Kv7 channels in airway smooth muscle may be important new targets for treatment of airway diseases such as COPD or asthma. Combination therapy with other bronchorelaxants may improve outcomes.
CONCLUSIONS
Acknowledgments:Lyuba BrueggemannChristina Robakowski Jennifer HaickShawn TateSamantha NeuburgDaniel Chiang
Leanne CribbsJulian Solway & Maria DowellMatthias Majetschak & Abhi Tripathi
Robert Love, Chris Wigfield, Jeffrey Schwartz
National Heart, Lung, & Blood Institute
30pM V1aAntag
Verap CalphC
Ro 31-8220
10nM Verap
Vaso
cons
tric
tion
as a
%
of M
axim
al O
uter
Dia
met
er
0
10
20
30
40
50
60
V1aAntag
Ro 31-8220
30 pM AVP
10 nM AVP
CalphC
Low [AVP] but not high [AVP] constrictor effectsdepend on PKC activation and L-type Ca2+ channels
KCNQ currents? Isolated Kv currents are not affected by blockers of other types of K+ channels.
Control Control 10 10 μμM Glibenclamide M Glibenclamide Washout 1 Washout 1 200 nM Iberiotoxin200 nM IberiotoxinWashout 2Washout 22 mM Tetraethylammonium2 mM TetraethylammoniumWashout 3Washout 32 mM 42 mM 4--aminopyridineaminopyridineWashout 4Washout 410 10 μμM LinopirdineM Linopirdine
1.01.0
0.60.6
0.20.2
2020--2020--4040--6060--8080V, mVV, mV
I, pA/pFI, pA/pF
I/II/Icc at at --20mV20mV
0.0
0.5
1.0
1.5
2.0
2.5
n=4 n=6 n=3 n=6
n=15n=6
n=11
* *
*
C IbTx Glib TEA 4-AP Linop XE-991 FlupMackie et al., JPET 2008
Celebrex® dilates mesenteric arterioles and enhances blood flow in vivo
B
20 m
control
13.90.5m
A AVP
9.70.4m
20 m
AVP + celecoxib
16.40.3m
20 m
bloo
d flo
w, 1
0-3m
m3 /s
ec
0.0
0.1
0.2
0.3
0.4
control AVP AVP+celecoxib
***
n=11 n=5 n=11
Heteromeric KCNQ4/5 channels in mesenteric artery myocytes.
Brueggemann et al. JBC 2014
PLA
sig
nal/c
ell
KCNQ4-KCNQ5
KCNQ4-TRPC6
0
10
20
30
40
*
KCNQ4-KCNQ5
KCNQ4-TRPC6
Ab ctrl.
phase PLA DAPI
Adv-GFPControl
50 µm50 µm
*
dnKCNQ5, n=6
control non-fluorescent, n=9control GFP, n=6
dnKCNQ4, n=5
***
V, mV-80 -60 -40 -20 0
I, pA/pF
0.4
0.6
*
Brueggemann et al. JBC 2014