Kv7 CHANNELS IN SMOOTH MUSCLE AS THERAPEUTIC …...Kv7 CHANNELS IN SMOOTH MUSCLE AS THERAPEUTIC...

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


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


KCNQ5 is expressed in A7r5 cells.


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


Ca2+ Spikes

PLD

PKC

CaL

K+


∆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


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


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



6,593,349 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


Ca2+ Influx

PLD

PKC

CaL

K+


∆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.


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


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

Kv7 CHANNELS IN SMOOTH MUSCLE AS THERAPEUTIC …...Kv7 CHANNELS IN SMOOTH MUSCLE AS THERAPEUTIC...

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