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Cardiovascular Research 62 (2004) 9 – 33
www.elsevier.com/locate/cardiores
Review
Pharmacology of cardiac potassium channels
Juan Tamargo *, Ricardo Caballero, Ricardo Go´mez, Carmen Valenzuela, Eva Delpo´n
Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain
Received 7 August 2003; received in revised form 11 December 2003; accepted 29 December 2003
Time for primary review 26 days
Abstract
Keywords: K+ channels; Cardiomyocytes; Pharmacology; Cardiac arrhythmias; Antiarrhythmic agents; Torsades de pointes
1. Introduction
Cardiac K+ channels determine the resting membrane
potential, the heart rate, the shape and duration of the action
potential and are important targets for the actions of neurotransmitters, hormones, drugs and toxins known to modulate
cardiac function [1 –3]. Blockers of certain or most K+
channels prolong the cardiac action potential duration
(APD) and refractoriness without slowing impulse conduction, i.e. they exhibit Class III antiarrhythmic actions, being
effective in preventing/suppressing re-entrant arrhythmias.
Unfortunately, drugs that delay the repolarization prolong
the QT interval of the electrocardiogram and represent a
major cause of acquired long QT syndrome (LQTS) [4– 6].
* Corresponding author. Tel.: +34-91-3941472; fax: +34-91-3941470.
E-mail address: [email protected] (J. Tamargo).
Cardiac K+ currents can be distinguished on the basis of
differences in their functional and pharmacological properties. In mammalian cardiac cells, K+ channels can be
categorized as voltage-gated (Kv) and ligand-gated channels
[1 –4]. The first category includes the rapidly activating and
inactivating transient outward current (Ito1), the ultrarapid
(IKur), rapid (IKr) and slow (IKs) components of the delayed
rectifier and the inward rectifier (IK1), whereas the ligandgated channels include those activated by a decrease in the
intracellular concentration of adenosine triphosphate (KATP)
or activated by acetylcholine (KAch) [1 –4]. The configuration and duration of the cardiac action potentials vary
considerably among species and different cardiac regions
(atria vs. ventricle) and specific areas within those regions
(epicardium vs. endocardium). This heterogeneity mainly
reflects differences in the type and/or expression patterns of
the K+ channels that participate in the genesis of the cardiac
action potential. Moreover, the expression and properties of
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2003.12.026
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Cardiac K+ channels are membrane-spanning proteins that allow the passive movement of K+ ions across the cell membrane along its
electrochemical gradient. They regulate the resting membrane potential, the frequency of pacemaker cells and the shape and duration of the
cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters and hormones and
class III antiarrhythmic drugs that prolong the action potential duration (APD) and refractoriness and have been found effective to prevent/
suppress cardiac arrhythmias. In the human heart, K+ channels include voltage-gated channels, such as the rapidly activating and inactivating
transient outward current (Ito1), the ultrarapid (IKur), rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward
rectifier current (IK1), the ligand-gated channels, including the adenosine triphosphate-sensitive (IKATP) and the acetylcholine-activated
(IKAch) currents and the leak channels. Changes in the expression of K+ channels explain the regional variations in the morphology and
duration of the cardiac action potential among different cardiac regions and are influenced by heart rate, intracellular signalling pathways,
drugs and cardiovascular disorders. A progressive number of cardiac and noncardiac drugs block cardiac K+ channels and can cause a marked
prolongation of the action potential duration (i.e. an acquired long QT syndrome, LQTS) and a distinct polymorphic ventricular tachycardia
termed torsades de pointes. In addition, mutations in the genes encoding IKr (KCNH2/KCNE2) and IKs (KCNQ1/KCNE1) channels have
been identified in some types of the congenital long QT syndrome. This review concentrates on the function, molecular determinants,
regulation and, particularly, on the mechanism of action of drugs modulating the K+ channels present in the sarcolemma of human cardiac
myocytes that contribute to the different phases of the cardiac action potential under physiological and pathological conditions.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
10
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
Fig. 1. Cardiac action potential (A) and the schematic representation of the
major ionic currents (B) contributing to its waveform. The amplitudes of the
depolarizing (downward) and repolarizing (upward) currents are not on the
same scales. Abbreviations: see text. Modified from Ref. [1].
arrhythmias and as potential targets of potentially useful
therapeutic agents. For more detailed review of structure –
function relationship, distribution and pharmacological
modulation of K+ channels, the reader is referred to recent
reviews [11 – 16].
2. K+ channel diversity and classification
K+ channels are formed by coassembly of pore-forming
a-subunits which surround a water-filled pore and accessory
h-subunits. Over 80 human K+ channel-related genes have
been cloned and characterized [1 – 4,11]. The genes encoding the a- and h-subunits of cardiac K+ channels are shown
in Table 1. Key features of a-subunits are: (a) a pore region
through which K+ ions flow across the plasma membrane,
(b) a selectivity filter that allows K+ but not other ions to
cross the pore, and (c) a gating mechanism that controls
switching between open-conducting and closed-nonconducting states and determines whether permeation occurs
in response to either changes in membrane potential or a
ligand. The recent identification of the atomic structures of
several K+ channels (KcsA, KvAP and MthK) provides the
basis for understanding, at a molecular level, the mechanisms that control ion selectivity and conduction [15]. K+
channels have been classified into three major families on
the basis of the primary amino acid sequence of the asubunit [1 –4,11]:
(1) Channels containing six transmembrane segments
and one pore-forming region (Fig. 2A). This architecture
is typical of Kv channels, whose members include Shakerrelated channels (Kv1 – 4), ether-a-go-go-related channels
(ERG) and KCNQ channels. Immediately after depolarization Kv channels move from the closed to the open state
(activation) and then many channels enter into a nonconducting state (inactivation), leading to a decline in activated
macroscopic current. When the membrane is repolarized
channels recover from the inactivated state and are once
again capable of opening in response to membrane depolarization. There are two major types of inactivation [16]. Ntype inactivation results from the occlusion of the intracellular mouth of the pore by a ‘‘ball and chain’’ mechanism
when the channel opens. C-type inactivation appears to
involve a rearrangement of residues in the external mouth
of the channel that becomes occluded.
Kv channel a subunits have six transmembrane-spanning
segments (S1 – S6) with cytoplasmic N- and C-terminal
domains and a pore loop between S5 and S6 bearing the
K+ selectivity filter signature TxGYG [1,4,11]. The pore
region, formed by S5 and S6 segments and the S5 – S6
linker, is responsible for K+ ion conduction and selectivity.
The S4 segment contains positively charged residues (R/K)
at approximately every third position and serves as the
voltage sensor. Most Kv channels contain a Pro-X-Pro
sequence (PXP) that induces a sharp bend in the S6 helices
near the activation gate and reduces the inner vestibule of
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K+ channels are not static but are influenced by heart rate,
neurohumoral state, pharmacological agents, cardiovascular
diseases (cardiac hypertrophy and failure, myocardial infarction) and arrhythmias (atrial fibrillation-AF) [1,7 –10].
The cardiac action potential reflects a balance between
inward and outward currents. Fig. 1 shows the relationship
between a cardiac action potential and the time course of
individual ionic currents that participate in its genesis. The
initial upstroke of the atrial and ventricular cells (phase 0) is
due to the activation of the fast inward Na+ current (INa).
Initial rapid repolarization (phase 1) is a consequence of the
rapid voltage-dependent inactivation of INa and the activation of Ito1 and the IKur. During phase 2 inward depolarizing
currents through Na+ (slowly inactivated) and L-type Ca2 +
channels (ICaL) are balanced by the different components of
the delayed rectifier K+ current (IKur, IKr and IKs). The
terminal phase 3 of repolarization is due to the increasing
conductance of the IKr, IKs and IK1. IK1 is also responsible
for the maintenance of the resting potential.
In this review we shall attempt to address (1) the function
and molecular basis of cardiac K+ channels, (2) the pharmacological modulation and its cellular consequences, and
(3) the signaling pathways involved in the regulation of
channel function and expression under pathophysiological
conditions. This information should provide fundamental
understanding of the role of K+ channels in modulating
cardiac function, in the genesis and maintenance of cardiac
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
11
Table 1
Genes encoding the a and h subunits of cardiac potassium channelsa
Current
a-Subunit
h-Subunit
Gene
Human
chromosomal
location
Name
Gene
Human
chromosomal
location
IKs
IKr
Kv7.1 (KVLQT1)
Kv11.1 (HERG)
KCNQ1
KCNH2
11p15.5
7q35 – 36
IKur
Kv1.5 (HK2)
KCNA5
12p13.3
minK
minK
MiRP1
Kvh1 (Kvh3)
Kvh2
KCNE1
KCNE1
KCNE2
KCNAB1
KCNAB2
21q22.1 – q22.2
21q22.1 – q22.2
21q22.1
3q26.1
1p36.3
IK1
Kir2.1 (IRK1)
Kir2.2 (IRK2)
Kir3.1 (GIRK1)
Kir3.4 (GIRK4)
Kir6.2 (BIR)
Kv4.3
Kv1.4
Kv4.1
Kv4.2
K2P1.1 (TWIK-1)
K2P2.1 (TREK1)
K2P3.1 (TASK-1)
K2P5.1 (TASK-2)
K2P6.1 (TWIK-2)
K2P9.1 (TASK-3)
K2P10.1 (TREK-2)
K2P13.1 (THIK-1)
K2P17.1 (TASK-4)
KCNJ2
KCNJ12
KCNJ3
KCNJ5
KCNJ11
KCND3
KCNA4
KCND1
KCND2
KCNK1
KCNK2
KCNK3
KCNK5
KCNK6
KCNK9
KCNK10
KCNK13
KCNK17
17q23.1 – 24.2
17p11.1
2q24.1
11q23
11p15.1
1p13.2
11p14.3 – 15.2
Xp11.23
7q31
1q42 – 43
1q41
2p24.1 – 23.3
6p21
19q13-1
8q24-3
14q31
14q24.1 – 24.3
6p21.1-2
SUR2A
KChIP2
ABCC9
KCNIP2
12p12.1
10
KChIP1
KChIP2
KCNIP1
KCNIP2
5q35
10
IKAch
IKATP
Ito1
K2P
a
Synonyms are those approved by the Human Genome Nomenclature-HGNC database (http://www.gene.ucl.ac.uk/nomenclature) and the International
Union of Pharmacology (http://www.iuphar-db.org/iuphar-ic/).
the pore [16]. Kv channels can be formed from four
identical a-subunits (homomultimers) or from combinations
of different a-subunits from the same subfamily (heteromultimers). In the Kv1 subfamily, tetramerization appears to be
controlled to some extent by the N-terminal intracellular T1
domain (T1) that immediately precedes S1. However, in
HERG and KCNQ1 channel subfamilies, C-terminal
domains appear to be important for heterotetramerization
[13,16].
(2) Inward rectifier K+ channels (Kirs) contain two
transmembrane domains (M1 and M2) connected by a pore
containing the G(Y/F)G sequence and intracellular N- and
C-termini (Fig. 2B). This architecture is typical of K1, KATP
and KAch channels [1– 3,17– 19]. They conduct K+ currents
more in the inward direction than the outward and play an
important role in setting the resting potential close to the
equilibrium potential for K+ (EK, approximately 90 mV
for [K+]o = 5 mM) and in repolarization [1,11,17 – 19]. Kir
channels form either homo- or heterotetramers.
(3) Four transmembrane segments and two pore channels
(K2P) have intracellular N- and C-termini and exist as
homodimers or heterodimers (Fig. 2C) [20 – 22]. The conventional G(Y/F)G of K+-selective motif is preserved in the
first pore loop, but it is replaced by G(F/L)G in the second.
K2P currents display little time- or voltage-dependence and
I/V curves can be described by the Goldman– Hodgkin –
Katz (GHK) equation, thus regulating resting membrane
potential and excitability. There are four classes of cardiac
K2P: TASK, TWIK, TREK and THIK.
The diversity of K+ currents in native tissues exceeds the
number of K+ channel genes identified. The explanations for
this diversity include alternative splicing of gene products,
post-translational modification, heterologous assembly of asubunits within the same family and assembly with accessory h-subunits that modulate channel properties (see below) [1– 4,11,23].
2.1. Auxiliary subunits
Recapitulation of the physiological features of the native
K+ current frequently requires accessory h-subunits. Most
Kvh subunits assemble with a-subunits giving rise to an
a4h4 complex. K+ channel h-subunits represent a diverse
molecular group, which includes cytoplasmic proteins
(Kvh1-3, KChIP and KChAP) that interact with the intracellular domains of Kv channels, single transmembrane
spanning proteins, such as minK and minK-related proteins
(MiRPs) encoded by the KCNE gene family, and large ATPbinding cassette (ABC) transport-related proteins, such as
the sulfonylurea receptors (SUR) for the inward rectifiers
Kir6.1 – 6.2 (Fig. 2) [2 –4,23 – 25]. Coexpression of h subunits with a-subunits regulates cell surface expression,
gating kinetics and drug-sensitivity of K + channels
[25,26]. Kvh1.1 – 1.3 and Kvh3.1 have an inactivation
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Name
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J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
domain at the N-terminus similar to the N-terminal inactivation ball of the a-subunit and when coassembled can
produce a N-type inactivation [26]. Kvh2.1 and Kvh4.1
behave as chaperone proteins, promoting proper protein
folding and/or subunit coassembly, channel trafficking and
cell surface expression of coexpressed subunits [26].
KChIPs are cytosolic Ca2 +-binding subunits that interact
with the N-terminus of the Kv4.3 a-subunits and enhance
their expression in the cell membrane [24,26]. Coexpression
of KChAP with Kv channels also enhances total current
density, suggesting a true chaperone function [23].
3. Cardiac voltage-gated channels
3.1. The transient outward current, Ito1
Ito is rapidly activated and inactivated in response to
depolarization [3,27]. Ito is the sum of a voltage-dependent,
4-aminopyridine (4-AP)-sensitive, calcium-independent K+
current (Ito1) and a 4-AP-insensitive, Ca2 +-activated Cl or
K+ current (Ito2). In human atrial and ventricular myocytes
the presence of Ito2 has not been clearly demonstrated, so it
will be not discussed further. Ito1 is responsible for early
rapid repolarization (phase 1) and determines the height of
the early plateau, thus influencing activation of other currents that control repolarization, mainly ICaL and the delayed
rectifier K+ currents (IK). Furthermore, variations in cardiac
repolarization associated with Ito1 differences strongly influence intracellular Ca2 + transient by modulating Ca2 +
entry via ICaL and Na+ – Ca2 + exchange, potentially exacerbating impaired Ca2 + cycling in heart disease [28].
Ito1 density is 4- to 6-fold higher in atrial tissue, Purkinje
fibers, epicardial and midmyocardial (M) cells than in the
endocardial cells [29,30]. In human subendocardial cells Ito1
is lower and recovers more slowly than in epicardial or M
cells [29]. In canine right atria, Ito1 density is lower in the
appendage than in the crista terminalis, pectinate muscle and
AV ring area cells [31]. Ito1 density is higher in the right than
in the left canine ventricular M cells [32] and differences in
Ito1 density across the ventricular wall are causally linked to
the J wave of the ECG [30]. The prominent epicardial Ito1
contributes to the selective electrical depression of the
epicardium during ischemia and to the development of a
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Fig. 2. Side view of the topology of three classes of K+ channels subunits. (A) Schematic representation of a voltage-gated K channel a subunit composed of
six membrane-spanning a helices (S1 to S6) and one pore. The C-terminal cytoplasmic domain is drawn off to the side of the T1 domain, but its precise relative
location is until yet unknown. The schematic representation of single transmembrane spanning proteins, such as minK and minK-related proteins (MiRPs) is
also shown. NBD: nucleotid binding domain (B) Schematic representation of the a subunit of an inward rectifier K+ channel (Kirs) formed only by two
transmembrane domains (M1 and M2) connected by a pore. The topology of the sulfonylurea receptor (SUR) that interacts with Kir6.x a subunits is also
shown. SUR has been proposed to have three transmembrane domains (TMDO, TMD1A and TMD2), each of which consists of five, five and six membrane
spanning regions. (C) Schematic representation of the a subunit of ‘‘background’’ channels formed by four transmembrane segments and two pores. SID:
self-interacting domain. Abbreviations: see text.
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
13
Table 2
Pharmacological blockade of voltage-gated potassium channels
Drug
Ito1
IKur
Kv1.5
4-AP
1.15 mM
50 AM
180 AM
Almokalant
Ambasilide
23 AM
34 – 45 AM
Amiodarone
4.9 AM
Azimilide
60%,
100 AM
Bepridil
Benzocaine
Bupivacaine
Candesartan
7%,
10 AM
39%,
100 AM
Chlorpheniramine
Chromanol 293B
Cibenzoline
E-4031
Ebastine
Fluvoxamine
HMR1556
Ibutilide
Imipramine
Indapamide
Irbesartan
Ketoconazole
L-735821
21.2 AM
140nM
0.5 AM
1 – 3 AM
115 AM
30 AM
259 AM
37%,
1 AM
>50 AM
>50 AM
13%,
3 AM
14%,
1 AM
101 AM
>10 AM
3.9 – 31 nM
3 AM
15%,
1 AM
10 nM
0.14 AM
< 10 AM
5.6 AM
0.55 AM
10 AM
18 AM
13%,
0.1 AM
104 AM
39%,
0.1 AM
8.4 AM
21 AM
6.5 nM
150 nM
7.2 AM
17.3 AM
27 AM
7.2 AM
10 – 110 nM
30%,
1 AM
0.17 AM
0.4 AM
25%,
1 AM
3.9 AM
1 mM
11%,
10 AM
>10 AM
1 AM
39%,
30 AM
>10 AM
50 AM
2 – 10 AM
>200 AM
>100 AM
130 AM
>0.1 mM
10 AM
6.5%,
1 AM
>100 AM
0.8 AM
18%,
1 AM
–
3.8 AM
25%,
10 AM
36%,
10 AM
>10 AM
98 AM
>10 AM
16 nM
39 AM (IK)
3.7 AM
34 nM
20 nM
3.4 AM
138 AM
20 nM
107 AM
1.2 AM
9%,
1 AM
193 AM
49 AM
28%,
1 AM
0.9 AM
173 nM
23%,
1 AM
19 nM
100 AM
40 nM
>30 AM
34 nM
1.3 AM
81 AM
4.75 AM
120 nM
15%,
1 AM
87 – 100 AM
314 AM
100%,
100nM
Loratadine
Losartan
LY97241
Mexiletine
MK-499
Nicotine
Nifedipine
NIP-142
0.7 – 5 AM
24 AM
Eprosartan
Flecainide
0.3 – 7 AM
22%,
1 AM
37 nM
Species
References
HAM,Ltk,
MEL
RVM
HAM,GPV,
CAM
RTV,RVM,
TXO,
HEK
HAM,GPV,
TXO
HEK,COS
Ltk
RTV,Ltk,CHO
CHO,Ltk
[38,76 – 78]
GPV,CHO
[135]
GPV,RVM
CV,TXO
[5,44]
[45,136]
GPV,TXO
GPV,HVM, COS
GPV
[5,44]
[46,195]
[137]
HEK,GPV
GPV,Ltk,TXO,RTV
HEK
COS,MEL
GPV,TXO
RTV,CHO
GPV,TXO
GPV
GPV,Ltk,CHO
[85,138]
[5,47,76]
[139]
[5,78,133]
[5]
[48,140]
[12,71,122,141]
[142]
[86]
AT-1,HAM,GPV
GPV,TXO,Ltk
[39,71,123]
[44,87]
CHO,Ltk
[84]
GPV,HAM,CNA,
HEK,MEL
HEK
GPV,TXO
[38,78,80,
137,143]
[144]
[49]
GPV,AT-1
RAM,GPV,CHO
[5,145,146]
[5,50,147]
GPV,CNA
CHO,Ltk
TXO
RVM,TXO
[51,188]
[88]
[148]
[190,191]
Ltk
GPV,Ltk,CHO
[89,149]
[86]
TXO,RTV
GPV
TXO
CNV,TXO
MEL,HEK,RTV
HEK
[5,47,150]
[137]
[129]
[52]
[53,78]
[90]
[5,12]
[39,40,80]
[41,81,130,131]
[42,128,132,194]
[81,133]
[82]
[43,83,134]
[84]
(continued on next page)
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Cisapride
Clofilium
Cocaine
Diltiazem
Diphenhydramine
Disopyramide
Dofetilide
Dronedarone
E-3174
68%,
10 AM
100%,
30 AM
15 nM
1 AM
KCNQ1/
KCNE1
32 AM
9.8 – 38 AM
31.6%,
50 AM
0.1 mM
1 – 3 AM
(IK)
1.1 AM
4%,
10 AM
IKs
2.8 AM
6.6 AM
901 AM
6.6 – 8.9 AM
21%,
0.1 AM
22 AM
HERG
50 nM
5.6 AM
Canrenoic acid
Cetirizine
Chloroquine
IKr
14
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
Table 2 (continued)
Drug
Ito1
Papaverine
Pentobarbital
Pimozide
IKur
Kv1.5
>100 AM
43 AM
Propafenone
4.8 AM
Quinidine
5 – 10 AM 5 AM
Risperidone
Ropivacaine
RP58866
Sematilide
Sertindole
D-Sotalol
Spironolactone
TEA
Tedisamil
Terfenadine
>30 AM
6 AM
80 AM
2.3 AM
>0.5 mM
2.1 AM
0.5 – 1 mM 1 mM
>10 mM
4.5 – 6 AM
>1 AM
1.1 AM
2.2 AM
IKs
KCNQ1/
KCNE1
2.75 mM
1.6 mM
18 nM
0.2 mM
0.2 mM
27%,
10 AM
0.76 AM
0.5 AM
(IK),
20%,
5 AM
9 nM – 2 AM 0.3 – 1 AM
30 – 260 nM 167 nM
20.3 AM
7 AM (IK)
22 nM
12 AM
14 nM
88 AM
23 AM
43%,
5 AM
0.2 AM
>100 AM
330 mM
>2 AM
>30 AM
HERG
2.5 AM (IK)
50 nM
9 – 350 nM
10 AM
31 nM
1.2 AM
0.25 AM
191 nM
>10 AM
14 AM
100 AM
45 AM
0.14 – 0.83 AM
Species
References
HAM,Ltk
GPV,TXO
HEK,CHO
[91]
[151]
[92]
HAM,GPV,Ltk,
RAM,CHO
[54,55,93,
152,153]
c 50 AM CHO,GPV,
AT-1,HAM,
TXO,Ltk,
CNA,HEK
RVM,CHO
Ltk,CHO
GPV,CNV
GPV
HEK
GPV,HAM,MEL
CHO
CNA,Ltk,MEL
GPV,RTV
Ltk,GPV,
TXO,RTV
RTV,GPV,TXO
GPV,CHO
GPV
>100 AM RTV,HEK,
COS,CNA
[12,38,80,94,
122,143,154]
[56,155]
[95,134]
[57,156]
[146]
[157]
[39,71,78,141]
[135]
[72,76,78]
[58]
[5,44]
[5,59,156]
[5,155]
[5]
[53,80,133,158]
Data are expressed as IC50 values (concentrations producing 50% inhibition of the current).
4-AP: 4-aminopyridine, AT-1: mouse atrial tumor cells, CHO: Chinese hamster ovary cells, CNA: canine atrial myocytes, CNV: canine ventricular myocytes,
COS: African green monkey kidney cells, CV: cat ventricular myocytes, GPV: guinea pig ventricular myocytes, HAM: human atrial myocytes, HEK: human
embryonic kidney cells, IK: delayed rectifier current, Ltk: mice fibroblasts, MEL: mouse erythroleukemia cells, RAM: rabbit atrial myocytes, RVM: rabbit
ventricular myocytes, RTV: rat ventricular myocytes, TEA: tetraethylammonium, TXO: oocytes of Xenopus laevis.
marked dispersion of repolarization between normal and
ischemic epicardium and between epicardium and endocardium, thereby providing the substrate for reentrant arrhythmias [33]. Furthermore, flecainide-induced abolition of the
action potential dome in epicardial but not in endocardial
cells leads to a marked dispersion of repolarization across
the ventricular wall that, when accompanied by prominent
conduction delays related to Na+ channel blockade, results
in extrasystolic activity through a phase 2 reentrant mechanisms [34].
3.1.1. Molecular basis of Ito1
Kv4.3 channels are the leading candidate for encoding
human Ito1 [35,36]. Kv4.3 mRNA decreases in failing hearts
and this reduction correlates with the reduction in Ito1
density, indicating that part of the downregulation of this
current may be transcriptionally regulated [3,9,35]. However, Kv1.4 channels possibly represent the Ito1 in the human
endocardium [3,29]. Heterologous expression of hKv4.3,
Kv1.4 or both does not reproduce the properties of the
native Ito1 [3,27,29]. KChIP2, when coexpressed with
hKv4.3, increases surface channel density and current
amplitude, slows the inactivation, accelerates the recovery
from inactivation and shifts the half-maximal inactivation to
more positive potentials [23,24]. Thus, features of Kv4.2/
KChIP2 currents closely resemble those of Ito1. In human
ventricle KChIP2 mRNA is 25-fold more abundant in the
epicardium than in the endocardium, and this gradient
parallels the gradient in Ito1 expression, while Kv4.3 mRNA
is expressed at equal levels across the ventricular wall. Thus,
transcriptional regulation of the KChIP2 gene is the primary
determinant of Ito1 expression in the ventricular wall [37].
3.1.2. Pharmacology of Ito1
Ito1 is blocked by 4-AP in the mM range in a fashion
suggesting a preferential interaction with the closed state
[38]. Table 2 shows the blocking potencies, expressed as
IC50 values (i.e, concentrations producing 50% inhibition of
the current), of several drugs on native Ito1 [38 –59]. 4-AP,
quinidine and propafenone produce an open channel blockade and accelerate Ito1 inactivation [38]. Quinidine, but not
flecainide, propafenone or tedisamil, produces a frequencydependent block of Ito1 that results from a slow rate of drug
dissociation from the channel [38,60]. Ambasilide reduces
Ito1 by changing conductance rather than by influencing the
gating mechanism [40].
It has been proposed that Ito1 blockers prolong the APD
in human atrial myocytes [61] and in ischemic ventricular
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Terikalant
Thioridazine
Triamterene
Verapamil
>10 AM
29%,
10 AM
4.4 AM
IKr
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
muscles [62]. However, since the net effects of Ito1 blockade
on repolarization may depend on secondary changes in other
currents, the reduction of Ito1 density can result in a
shortening of the ventricular action potential [27,62,63].
Moreover, heterogeneous ventricular distribution of Ito1 may
increase the disparity in the APD and facilitate the initiation
or perpetuation of reentrant arrhythmias [64]. Thus, the role
of Ito1 in the control of human cardiac repolarization still is
unclear.
3.1.4. Regulation of Ito1
The phosphorylation, mediated by protein kinase A
(PKA) and C (PKC), alters channel properties by modifying
the kinetics and/or the expression of active channels present
on the cell surface. Phorbol 12-myristate 13-acetate (PMA),
an unspecific activator of serine/threonine and tyrosine
protein kinases, suppresses both Kv4.2 and Kv4.3 currents
and native Ito1 but not after preincubation with PKC
inhibitors (chelerythrine) and staurosporine, a nonspecific
inhibitor of serine/threonine protein kinases [68]. PKC
increases the inactivation time constants and slows the time
course of recovery from inactivation of Ito1. a-adrenergic
agonists (phenyleprine and methoxamine) reduce Ito1 in rat
ventricular myocytes [63], whereas h-adrenergic agonists
have no effects on Ito1. Furthermore, phenylephrine and
carbachol inhibit Kv4.3 currents only when coexpressed,
respectively, with a1-adrenoceptors or M1 receptors and
this effect is also prevented by chelerythrine, suggesting that
it may be mediated by activation of PKC [68]. In hKv4.1
channels, PMA causes an initial increase followed by a
reduction in current amplitude and these effects are prevented by staurosporine or chelerythrine [69]. Angiotensin
II inhibits Ito1 in rat ventricular myocytes, an effect pre-
vented by PD123391, a selective antagonist of AT2 receptors. This inhibition is abolished by okadaic acid, an
inhibitor of protein phosphatases 1 and 2A, suggesting that
the stimulation of AT2 receptors activates the serine –threonine protein phosphatases to reduce Ito1 density [70].
3.2. The delayed rectifier K+ currents
In the human heart, IK can be separated into at least three
different components, the ultrarapid (IKur), rapid (IKr) and
slow (IKs) components. These currents exhibit different
kinetics and pharmacological properties, are regulated by
different intracellular signaling pathways and encoded by
separate genes [1– 4,71 –75].
3.2.1. The ultrarapid component of the delayed rectifier K+
current, IKur
Atrial cells possess a K+ current that activates rapidly in
the plateau range (s < 10 ms), displays outward rectification
and inactivates very slowly during the time course of the
action potential [72,76,77]. IKur has been recorded in human
atria but not in human ventricular tissue, so that it is the
predominant delayed rectifier current responsible for human
atrial repolarization [72,77]. IKur density is similar in cells
isolated from different regions of the canine right atrium
[31]. Kv1.5 protein is located at the intercalated disks, a
pattern that is disrupted after ischemic damage.
3.2.1.1. Molecular basis of IKur . Kv1.5 (613 amino acids)
encodes the a-subunit of the IKur channel (10 – 14 pS).
Heterologous expression of Kv1.5 results in a delayed
rectifier current with the biophysical and pharmacological
characteristics of IKur and exposure to Kv1.5 antisense
oligodeoxynucleotides-AsODN specifically inhibits IKur
density in human atrial myocytes [72,77]. Kv1.5 subunits
coassemble with Kvh1.2 subunits to form the IKur in human
atrium [26].
3.2.1.2. Pharmacology of IKur . IKur is relatively insensitive
to TEA [76,78], Ba2 + and class III antiarrhythmics of the
methanesulfonanilide group [39,78], but is highly sensitive
to 4-AP [38,76 – 78]. Selective inhibition of IKur by 4-AP
prolongs the human atrial APD [77] and the atrial but not
the ventricular refractoriness in the dog [84]. The potency of
various drugs to block IKur and heterologously expressed
Kv1.5 channels is summarized in Table 2 [47,58,76 –95].
3.2.1.3. State specific block of IKur . Antiarrhythmic drugs
and local anesthetics are weak bases that predominate in
their cationic form at physiological pH. The cationic form
enters into the permeation pathway only after channel
opening and blocks the current by binding at a site in the
internal mouth of the ionic pore. The binding of these drugs
is determined by an electrostatic component reflecting the
electrical binding distance (about 20% of the transmembrane electrical field) and a hydrophobic component that
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3.1.3. Pathological role
Heart failure, cardiac hypertrophy and myocardial ischemia and infarction are associated with prolongation of the
APD, an effect that has been attributed, in part, to a downregulation of Ito1 [8,9,27]. The decrease in Ito1 in heart
failure may be adaptive in the short-term because increased
depolarization during the cardiac cycle means that more
time is available for excitation –contraction coupling, which
mitigates the decrease in cardiac output, but it becomes
maladaptive in the long-term, since a prolongation of the
APD may contribute to arrhythmogenesis, either by causing
inhomogeneous repolarization or by increasing the likelihood of early afterdepolarizations [27]. However, there is
also evidence of an up-regulation of Ito1 in hypertrophied
cardiac myocytes [9,65] and in cardiac myocytes after
induced myocardial infarction [8,66]. It can be speculated
that up-regulation of Ito1 may represent a protection mechanism counteracting the excessive prolongation of the APD
and Ca2 + influx, in order to minimize the incidence of
ventricular proarrhythmia. Chronic AF reduces Ito1 density
and Kv4.3 mRNA levels [10]. Hypothyroidism reduces the
expression of KCND2 (Kv4.2) genes [67].
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J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
largely determines the intrinsic stability of the drug – receptor complex, and thus, the affinity (Fig. 3A and B). It has
been proposed that most of the critical residues for hydrophobic binding of quinidine in the hKv1.5 channel are
aligned on one side of the putative S6, so that it is
hypothesized that this region is the pore-lining region and
is important in determining drug affinity and specificity
[96]. Stereoselective bupivacaine block of hKv1.5 channels
is determined by a polar interaction with T507 and two
hydrophobic interactions at positions L510 and V514 [97].
The length of the alkyl substituent at position 1 of bupivacaine-related local anesthetics determine their potency and
stereoselectivity, possibly because this alkyl chain interacts
with a hydrophobic residue (L510) in the S6 of the channel
[98]. In contrast, at low concentrations uncharged-neutral
(benzocaine [82,99], nifedipine [100], loratadine [89]) and
negatively charged drugs (angiotensin II type 1 receptor
antagonists [84,86,88]) affect the gating properties of
hKv1.5 channels without obvious block (Fig. 3C – E), and,
frequently, they increase the current amplitude at voltages at
which activation of channels reaches saturation (Fig. 3C)
[82,86]. Moreover, these drugs produce a voltage-dependent
block, but their voltage-dependence is exactly a mirror
image of that observed in the presence of cationic drugs
(Fig. 3D) [84,93]. Furthermore, the residues in the S6
segment that determine the binding of quinidine and bupivacaine also determine the binding of neutral and acid drugs
(benzocaine [99]) suggesting the existence of a common
receptor site at the channel level. Very recently ala-scanning
mutagenesis within the pore helix and the S6 segment
identifies the residues (T479, T480, V505, I508 and
V512) that determine the binding of S0100176, a new
selective open IKur blocker [101]. The implicated residues
face towards the central cavity and overlap with putative
binding sites for other blockers and Kv channels, like
HERG and KCNQ1, indicating a conservation of drug
binding sites among different K+ channel families.
The external mouth of the channel pore formed by the P
loop and adjacent S5 –S6 segments is the binding site for
some drugs [3,12]. Long-chain polyunsaturated fatty acids
(arachidonic and docosahexaenoic acids) bind to an external
site and block Kv1.5 channels [102] and R(+)-N-methylbupivacaine, a membrane-impermeant permanently charged
drug, produces different effects on Kv1.5 channels when
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Fig. 3. Effects produced by weak bases (bupivacaine and IQB-9302) and weak acids (losartan) on hKv1.5 channels. (A) Current traces of hKv1.5 currents
obtained by applying 250 ms depolarizing pulses from 80 to + 60 mV in 10 mV steps in control conditions and in the presence of bupivacaine (20 AM). Tail
currents were elicited upon repolarization to 40 mV. (B) Relative hKv1.5 current obtained in the presence and in the absence of bupivacaine and IQB-9302
(20 AM), plotted together with the activation curve (dotted line). At membrane potentials positive to 0 mV, a shallower increase in block was observed. This
voltage-dependence was fitted (continuous line) following a Woodhull equation ( f =[D]/{[D] + KD*exp( zdFE/RT)}) and yielded d-values of 0.20 and 0.17
for bupivacaine and IQB-9302, respectively. Modified from Ref. [83]. (C) Effects of losartan (1 AM) 4 min after beginning the infusion (left) and after steadystate was reached (right). hKv1.5 currents traces were elicited by 500 ms pulses to + 60 mV and tail currents upon return to 40 mV. (D) Relative hKv1.5
current obtained in the presence and in the absence of losartan, plotted together with the mean activation curve (dotted line). At membrane potentials positive to
0 mV, a shallower decrease in block was observed. This voltage-dependence was fitted (continuous line) following a Woodhull equation and yielded zy-values
of 0.27. (E) Effects of losartan on the voltage-dependence of hKv1.5 channels activation. Activation curve in control conditions was fitted with a single
Boltzmann component ( y = A/{1 + exp[(Vh Vm)/k]}) (continuous line). Dashed line shows that this approach was not optimal for data in the presence of
losartan, whereas best fit was obtained with a sum of two Boltzmann components. Modified from Ref. [86].
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
applied from the intra or the extracellular side of the
membrane, thus confirming the existence of an external
binding site for bupivacaine-like local anesthetics [103].
3.2.1.5. Regulation of IKur.. Membrane depolarization
and elevated [K+]o, reduce Kv1.5 expression, while
dexamethasone increases ventricular Kv1.5 mRNA [72].
Cyclic adenosine 3V,5V-monophosphate (cAMP), mechanical stretch and hyperthyroidism increase, whereas extracellular acidosis, phenylephrine, and hypothyroidism
decrease Kv1.5 expression [67,72,107]. Kv1.5 mRNA
levels decrease in hypertensive hypertrophied rat ventricle
[108] and in the epicardial border zone of the infarcted
canine ventricle [8].
hKv1.5 has one consensus site for phosphorylation by
PKC located on the extracellular S4 – S5 linker and 4
consensus sites for PKA located in the N- and C-terminal
domains. Isoproterenol increases IKur in human atrial myocytes and this effect is mimicked by direct stimulation of
adenylate cyclase and suppressed by a PKA inhibitor
peptide [109]. In the presence of propranolol, phenylephrine
inhibits IKur, an effect that is prevented by the PKC inhibitor
bisindolylmaleimide. These results indicate that h-adrenergic stimulation enhances, whereas a-adrenergic stimulation
inhibits IKur and suggest that these actions are mediated by
PKA and PKC, respectively.
Coexpression of Kv1.5 channels and human thrombin or
rat 5-HT1c receptors, two receptors that increase phospholipase C (PLC) activity, inhibits Kv1.5 current amplitude
without altering the kinetics or voltage sensitivity of activation [110]. Simultaneous injection of inositol 1,4,5-tri-
sphosphate and superfusion of PMA reproduces the
modulation of the Kv1.5 current suggesting that these
receptors can modulate Kv1.5 channels by increasing PLC
activity [110]. The hKv1.5 channel can associate via its Nterminus-located proline-rich sequences with the SH3 domain of the Src tyrosine kinase [111] and coexpression of
Kv1.5 with v-Src leads to tyrosine phosphorylation of the
channel and inhibition of the current.
Coexpression of Kvh1.3 with Kv1.5 induces a fast
inactivation and a hyperpolarizing shift in the activation
curve, i.e. Kvh1.3 subunit converts Kv1.5 from a delayed
rectifier with a modest degree of slow inactivation to a
channel with both fast and slow components of inactivation
[26]. These effects do not occur when Kv1.5 is coexpressed
with either Kvh1.2 or Kvh1.3 lacking the N-terminus, after
removal of a consensus PKA phosphorylation site on the
Kvh1.3 N-terminus (S24) or following preincubation with
calphostin C, a PKC inhibitor [26]. Moreover, okadaic acid
blunts the effects of calphostin as predicted if protein
dephosphorylation be required to remove the h-induced
inactivation. These data suggest that Kvh1.2 and Kvh1.3
subunit modification of Kv1.5 currents require phosphorylation by PKC or a related kinase. PMA has minimal effect
on Kv1.5 channels, but markedly reduces Kv1.5/Kvh1.2
currents and this effect is inhibited by chelerythrine, indicating that Kvh1.2 enhances the response of Kv1.5 to PKC
activation [112]. Kv1.5/Kvh1.3 channels are less sensitive
to quinidine- and bupivacaine-induced block than homomeric Kv1.5 channels [113]. These results suggest that the
Kvh1.3 subunit markedly reduce the affinity of these drugs
for their internal receptor site in hKv1.5 channels.
3.2.2. The rapidly activating component of the delayed
rectifier K+ current, IKr
IKr is characterized by rapid activation at 30 mV, rapid
inactivation and strong inward rectification at positive potentials which is due to rapid voltage-dependent C-type inactivation [16,71,73]. Inward rectification results from the fact
that channel inactivation develops faster than channel activation at positive potentials and limits the amount of time that
channels spend in the open state [73,114]. On repolarization
the rate of recovery from inactivation through the open state is
much more rapid than deactivation, which at voltages negative to 0 mV results in a large outward current and promotes
phase 3 of repolarization. Thus, IKr plays an important role in
governing the cardiac APD and refractoriness.
IKr has been identified in human atrial and ventricular
myocytes, rabbit sinoatrial (SA) and atrioventricular (AV)
nodal cells and Purkinje cells [3,14]. In rabbit SA myocytes
HERG channels play an important role in the pacemaker
activity and IKr blockers decrease the maximum rate of
diastolic depolarization [115]. In rats IKr density is higher
in atria than in ventricles, while in humans HERG expression
is higher in the ventricles [116]. In canine atria IKr density is
larger in the AV ring region and the left atrial wall than in
crista terminalis, pectinate muscles, appendage cells and right
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3.2.1.4. Clinical consequences of IKur block. IKur is a
promising target for the development of new safer antiarrhythmic drugs to prevent atrial fibrillation and/or flutter
without a risk of ventricular proarrhythmia [104]. However,
in patients with chronic AF, ICaL is inhibited which limits
the increase in the plateau height produced by IKur blockade
resulting in a significative lengthening of the atrial APD.
However, IKur density and Kv1.5 expression are reduced in
these patients [10], so that the degree of APD prolongation
produced by selective IKur blockers under these conditions is
unknown. In a rat model, rapid atrial pacing immediately
and transiently increases Kv1.5 mRNA levels (Kv4.3
mRNA levels decreased, while mRNA levels of Kv2.1,
erg, KCNQ1 and KCNE1 remained unaltered), which might
contribute, at least in part, to the rapid shortening of the
atrial refractoriness at the onset of AF [105]. If so, selective
IKur blockers might antagonize the shortening of the atrial
APD produced at rapid atrial rates and prevent episodes of
paroxysmal AF. Selective IKur blockers (NIP-142, S9947,
AVE0118, S0100176 and S20951) have no effect on the QT
interval and markedly prolong left vs. right atrial refractoriness, decrease left atrial vulnerability and reverse AF to
sinus rhythm [90,104,106]. Unfortunately, there are few
clinical data on the efficacy and safety of these drugs.
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J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
atrial wall, which may account for the shorter APD of the left
atria [14,31]. However, in the canine ventricle IKr is distributed homogeneously [117], while in guinea pig left ventricle,
IKr is smaller in subendocardial than in midmyocardial or
epicardial myocytes [118]. In rabbit left ventricle, IKr is
greater in the apex than in the basal regions and IKr blockers
cause more significant APD prolongation in the apex than in
the base, increasing the regional dispersion of APD [119].
3.2.2.2. Molecular basis of IKr . HERG a-subunits (1159
amino acids) encoded by the KCNH2 gene coassemble to
form heterotetrameric channels [120]. However, native IKr
and HERG channels expressed in heterologous systems
differ in terms of gating, regulation by external K+ and
single channel conductance [73,74]. These findings suggest
the presence of a modulating subunit that co-assembles with
HERG in order to reconstitute native IKr currents. A likely
candidate is MiRP1 (123 amino acids) encoded by the
KCNE2 gene. When coexpressed with HERG, KCNE2
shifts the HERG activation curve in the positive direction,
accelerates the rate of deactivation, decreases single channel
conductance (from 13 to 8 pS) and mediates the direct
stimulatory effect of cAMP on HERG/KCNE2 channels
[120,121]. KCNE2 reduces channel sensitivity to [K+]o and
enhances sensitivity to clarithromycin [120] but not to
dofetilide, E-4031 or quinidine [122]. KCNE2 can also
modulate KCNQ1, Kv4.2 and even the distinctly related
hyperpolarization-activated cation (HCN) pacemaker channels [74]. HERG can coassemble with other h-subunits like
minK encoded by KCNE1 gene (Table 1). Treating AT-1
cells with AsODNs against KCNE1 reduces IKr amplitude
[123] and when HERG and KCNE1 are coexpressed in
HEK293 cells, the HERG current amplitude increases.
3.2.2.3. Pharmacology of IKr . IKr is the target of class III
antiarrhythmic drugs of the methanesulfonanilide group
(almokalant, dofetilide, D-sotalol, E-4031, ibutilide, and
MK-499) (Table 2). These drugs produce a voltage- and
use-dependent block, shorten open times in a manner
consistent with open-channel block and exhibit low affinity
for closed and inactivated states [73,74,128,129]. Drugs that
block both native IKr and heterologously expressed HERG
channels are shown in Table 2 [6,12,71,122,123,129 – 158]
and in Fig. 4A and B. IKr blockers prolong atrial and
ventricular APD (QT prolongation) and refractoriness in
the absence of significant changes in conduction velocity
(AH, HV and PR intervals) [159]. In animal models, IKr
blockers suppress ventricular tachycardia induced by
programmed electrical stimulation or a new ischemic insult
in dogs with prior infarct [159,160]. However, these drugs
are probably not effective against triggered activity or
increased automaticity [159].
Some LQT2-causing HERG mutations give rise to defects
in channel trafficking resulting from improper protein folding and/or incorrect molecular assembly in the sarcoplasmic
reticulum and/or Golgi apparatus, resulting in their retention
and degradation by quality control machinery. Trafficking of
some mutant channels (N470D, G601S) into the sarcolemma
can be restored by HERG channel blockers (i.e. cisapride,
terfenadine, astemizole, E-4031) [73,126,127], even when
fexofenadine rescues mutant HERG channels at concentrations that do not cause channel block [127]. However, since
IKr blockers failed to rescue other trafficking-defective
mutants, it is evident that multiple mechanisms may exist
for pharmacological rescue of LQT2 mutations [127]. Moreover, some drugs (almokalant [12], norpropoxyphene [161],
azimilide [162], candesartan (Fig. 4C –E) [84] and E3174,
the active metabolite of losartan [86]) can exhibit an ‘‘agonist’’ IKr action at low concentrations. Flufenamic acid and
niflumic acid also increase HERG currents by accelerating
channel opening [163]. These results open the possibility of
developing new IKr openers for the treatment of patients with
congenital (LQT2) or drug-induced LQTS. Lysophosphatidylcholine increases HERG currents, an effect that may
contribute to K+ loss in the ischemic heart [164].
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3.2.2.1. Channel gating. The S4 –S5 linker and the Cterminal half of the S6 are crucial components of the
activation gate and point mutations at these regions have
marked effects on the voltage-dependence and kinetics of
channel activation and deactivation [73,74]. The N-termini
of HERG is involved in the slow deactivation process and
some mutations in this region linked with type 2 (LQT2) of
the Romano – Ward variant of the congenital long QT syndrome (cLQTS) reduce outward currents through the HERG
channel by accelerating channel deactivation [74]. Deletion
of amino acids 2 to 354 or 2 to 373 markedly increase HERG
deactivation rates [73]. HERG channels also contain a PAS
(Per –Arnt– Sim) domain on their cytoplasmic N-terminus
that may interact with other regions of HERG such as the
S4 – S5 linker to affect channel deactivation [73,74,114]. Ctype inactivation of HERG channels is slowed by extracellular TEA and elevated [K+]o and can be removed by
mutations in the outer mouth of the pore or the N-terminal
half of S6 [73,74]. It involves a rearrangement of the outer
pore region of the channel that either constricts the pore and
prevents ion fluxes, or alters the ion selectivity in such a way
that the channel cannot conduct K+ currents.
KCNH2 and KCNE2 have been identified as the loci of
mutations associated with LQT2 and LQT6 of the Romano– Ward variant of cLQTS, respectively [4,124,125]. The
cLQTS is a complex disease characterized by marked QTinterval prolongation and polymorphic ventricular tachycardias called torsades de pointes (TdP), causing syncope,
seizures and sudden death [4,6,124,125]. More than 100
mutations in the KCNH2 gene have been described, including frameshifts, insertions, deletions and missense and
nonsense mutations [124 – 127]. Mutant channels cause a
net reduction in outward K+ current during repolarization
that can result from different mechanisms, including generation of nonfunctional channels, altered channel gating and
abnormal protein trafficking.
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
19
An increasing number of drugs with diverse chemical
structures block IKr, delay ventricular repolarization, prolong the QT interval (acquired LQTS) and induce TdP (for
review see Refs. [5,6,165,166]). Thus, it is important to
understand the molecular determinants of drug binding to
HERG channels as well as the consequences of drug action
to develop safer and more effective drugs.
3.2.2.4. Structural basis of IKr/HERG blockade. Most IKr
blockers gain access to a binding site from the intracellular
side of the membrane and require channel opening before
they can gain access to a high affinity binding site located
inside the channel vestibule [73,74]. Once inside the pore, IKr
blockers bind within the central cavity of the channel between
the selectivity filter and the activation gate and can be trapped
in the inner vestibule by closure of the activation gate when
the channel deactivates during repolarization [167]. If a drug
is charged and appropriately sized, then block is nearly
irreversible as long as channels are not reopened even at
negative potentials. However, the drug-trapping hypothesis
predicts that unbinding and exit from the channel vestibule of
a positively charged drug should be favored by membrane
hyperpolarization if not impeded by the closed gate. Indeed,
using a mutant HERG channel (D540K) that opens in
response to hyperpolarization, it was found that channel
reopening allows recovery from block by MK-499 [177].
The finding that drugs with such different chemical
structures and sizes block IKr can be interpreted through
two unique structural features of the HERG channel: (a) the
lack of the PXP motif in the S6 segment (Fig. 2A) that
makes larger volume of the inner vestibule of the channel
pore thus increasing easy drug entry. Kv1 – 4 channels
contain the PXP sequence that is believed to induce a
sharp bend in the inner S6 helices of Kv channels [16]
reducing the inner vestibule and precluding the trapping of
large molecules like MK-499. (b) The presence of two
aromatic residues (Y652, F656) located in the S6 domain
facing the channel vestibule, that establishes specific interactions with aromatic moieties of several drugs by k
electron stacking [129]. Alanine mutagenesis of both residues drastically reduces the potency of channel block by
MK-499, dofetilide, quinidine, cisapride, terfenadine or
chloroquine [129,136,168]. Mutations that result in loss
of inactivation (S631A, G628C/S631C) reduce the affinity
of methanesulfonanilides, while mutations that increase
inactivation (T432S, A443S, A453S) enhance the blocking
potency of dofetilide [74,169]. It has been hypothesized
that the reduced affinity of noninactivating HERG mutant
channels is not due to inactivation per se but to inactivation
gating-associated reorientation of residues Y652 and F656
in the S6 domain that comprise a high-affinity binding site
[168].
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Fig. 4. Effects produced by propafenone (a weak base) and by candesartan (weak acid) on HERG channels. (A) HERG current traces obtained by applying 5 s
pulses from 80 to + 60 mV in 10 mV steps in control conditions and with 2 AM propafenone. Tail currents were elicited upon return to 60 mV (B)
Relative current as a function of the membrane potential. Block increases in the range of membrane potentials that coincides with the activation of HERG
channels. At membrane potentials positive to 0 mV, a shallower increase in block was observed. This voltage-dependence was fitted following a Woodhull
equation and yielded y-values of 0.09. Modified from Ref. [153]. (C) HERG current traces obtained with the voltage protocol illustrated at top for control
conditions and with candesartan. (D) Current – voltage relationships (5 s isochronal) of HERG channels recorded in the absence and in the presence of
candesartan (0.1 AM). (E) Effects of candesartan on the voltage-dependence of HERG channels activation. Activation curves in the absence and in the presence
of drug were fitted with a single Boltzmann component. Squares represent the fractional block ( f = IDRUG/ICON) from data obtained in the presence and in the
absence of candesartan. In panels D and E, *P < 0.05 vs. control. Modified from Ref. [84].
20
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
Very recently, the selective serotonin reuptake inhibitor
fluvoxamine has been shown to exhibit HERG channel
blocking properties that are different from those previously
described. In fact, the S6 mutations, Y652A and F656A, and
the pore helix mutant S631A only partially attenuate the
fluvoxamine-induced blockade at concentrations causing
profound inhibition of wild-type HERG channels [144].
Moreover, this blockade resembles that produced by canrenoic acid (CA), the main metabolite of spironolactone, on
HERG channels expressed in CHO cells [135]. Fluvoxamineand CA-induced HERG block is far more rapid (occurring
within 10 msec) than that produced by the more potent
methanesulfonanilides, suggesting that they exhibit either a
closed-state block or extremely rapidly developing open
channel blockade. Furthermore, channel inactivation was
not a prerequisite for fluvoxamine- and CA-induced block.
3.2.2.6. Regulation of IKr . HERG channels presents four
cAMP-dependent PKA phosphorylation sites and a cyclic
nucleotide binding domain (CNBD) in the C-terminal and
can be modulated via the cAMP-PKA phosphorylation
pathway [121,171]. Activation of h-adrenergic receptors
and elevation of intracellular cAMP levels regulate HERG
channels both through PKA-mediated effects and by direct
interaction with the protein [121]. PKA activation reduces
HERG current amplitude and induces a depolarizing shift in
the voltage-dependent activation curve [121,171]. This shift
is inhibited by specific PKA inhibitors (H89, KT5720) and
in channels missing all PKA phosphorylation sites (S283A,
S890A, T895A, S1137A), while coexpression of HERG
with KCNE1 or KCNE2 accentuates the cAMP-induced
voltage shift [121]. The net result of these effects is a
reduction in HERG current. However, isoproterenol
increases IKr in guinea pig ventricular myocytes, an effect
that was inhibited by bisindolylmaleimide [171]. In rabbit
SA cells isoproterenol increases IKr and this effect is
inhibited by H89, but not by bisindolylmaleimide [172].
These results suggest that regulation of IKr may be speciesand tissue-specific and may also depend strongly on the
experimental conditions.
HERG channels are modulated by PKC independently of
direct phosphorylation of the channel [173]. PMA causes a
positive shift of activation and reduces IKr and its effects can
still be observed when the PKC-dependent phosphorylation
sites are deleted by mutagenesis. Changes in phosphatidyl
4,5-biphosphate (PIP2) levels result from activation of
several adrenergic and muscarinic receptors. PIP2 increases
HERG current and shifts the voltage-dependence of activation in a hyperpolarizing direction [174]. Moreover, in cells
coexpressing the a1A-receptor and HERG, phenylephrine
reduces HERG currents and this effect is prevented by PIP2
but not by PKC inhibition, suggesting that the mechanism is
due to G-protein-coupled receptor stimulation of PLC
resulting in the consumption of endogenous PIP2. Since
there are several polycationic segments in the cytoplasmic
C-terminus of HERG, it is possible that an electrostatic
interaction between the negatively charged phosphate
groups of PIP2 and positively charged amino acids in the
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3.2.2.5. Clinical consequences of IKr block. IKr blockers
prolong atrial and ventricular APD and the QT interval and
may cause TdP that can degenerate into ventricular fibrillation and sudden cardiac death. Proarrhythmia induced by IKr
blockers is related to [4,5,165,166]: (a) excessive prolongation of APD near plateau voltages which favor the development of early after-depolarizations and (b) a more marked
prolongation of the APD in M-cells than in subepicardial or
subendocardial ventricular muscle possibly because of the
relative scarcity of IKs in M cells [117]. Thus, triggered focal
activity and ventricular reentry associated to an increased
inhomogeneity of repolarization across the ventricular wall
would lead to the development of TdP. The incidence of TdP
increases in the presence of hypokalemia, slow heart rates
(recent conversion from AF), QT prolonging drugs, preexisting cardiac diseases (hypertrophy, myocardial infarction), female gender or baseline QTc interval >0.46 s [4– 6].
Unlike most other K+ currents, IKr amplitude increases
upon elevation of [K+]o and decreases after removal of
extracellular K+ [73,74]. Elevation of [K+]o reduces C-type
inactivation and increases the single channel conductance of
HERG channels [154]. This explains why the APD are
shorter at higher [K+]o and longer at low [K+]o and the
association between hypokalemia, APD prolongation and
induction of TdP in patients treated with IKr blockers. On
the contrary, modest elevations of [K+]o using K+ supplements and spironolactone in patients given IKr blockers or
with LQT2 significantly shortens the QT interval and may
prevent TdP [170]. Moreover, the antiarrhythmic actions of
IKr blockers can be reversed during ischemia, which is
frequently accompanied by elevations of the [K+]o in the
narrow intercellular spaces and by catecholamine surges that
occur with exercise or other activities associated with fast
heart rates [159,160].
Unfortunately, the prolongation of the APD produced by
IKr blockers is more marked at normal resting potentials and
slow heart rates, while at depolarized potentials or during
tachycardia this prolongation is much less marked or even
absent [5,159,160]. This reverse use-dependence limits
antiarrhythmic efficacy, while maximizing the risk of TdP
associated with bradycardia-dependent early afterdepolarizations. In guinea pigs, reverse use-dependence is attributed
to a progressive IKs accumulation as the heart rate increased
(due to the incomplete deactivation of this current), which
shortens the APD and offsets the APD prolongation produced by the IKr blocker [73]. Another explanation is that
IKr block itself is reverse use-dependent, this property being
promoted by KCNE2 coexpression [120].
All these disadvantages, together with the finding that
inherited mutations of KCNH2 and KCNE2 genes are
linked to LQT2 and LQT6 forms of the Romano– Ward
syndrome, have decreased the initial interest for developing
selective IKr blockers.
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
channel protein can maintain the channel in an open state
[174]. In human ventricular myocytes, endothelin-1 inhibits
IKr, but fails to modify Ito1 and IK1 [175].
3.2.2.7. Pathological role. IKr and HERG currents are
unchanged in patients with chronic AF [10] and homogeneously distributed in failing canine hearts [176]. ATP,
derived from either glycolysis or oxidative phosphorylation,
is critical for HERG function. Both hyper- and hypoglycemia inhibit HERG currents and can cause QT prolongation
and ventricular arrhythmias [177]. IKr density and HERG
mRNA levels are reduced in myocytes from infarcted canine
ventricle [178]. However, IKr density increases in subendocardial Purkinje cells from the 48 h infarcted heart [8], an
effect that may increase the proarrhythmic effects of IKr
blockers in patients with myocardial infarction.
3.2.3.1. Molecular basis of IKs. IKs is generated by the
coassembly of four pore-forming KCNQ1 (676 amino
acids) and two accessory KCNE1 h-subunits [181]. KCNE1
(129 amino acids) exhibits a single transmembrane spanning
domain; the N-terminus is extracellular and the C-terminus
intracellular. Heterologous expression of KCNQ1 produces
rapidly activating, slowly deactivating currents, while
KCNE1, by itself, does not form functional channels.
Coexpression of KCNQ1/KCNE1 slows activation and
deactivation kinetics, shifts the voltage dependence of
channel activation to more positive potentials and increases
the macroscopic current amplitude, thus reproducing the
biophysical properties of the native cardiac IKs [182].
Additionally, KCNE1 confers regulation of IKs by PKC
and alters the pharmacology of KCNQ1 [74,183].
KCNE1 is abundant in the SA node, but less abundant
although homogeneously distributed throughout the ventricular wall and in the mouse heart expression is largely
restricted to the conducting system. An alternatively spliced
variant of KCNQ1 with a N-terminal deletion that produces
a negative suppression of KCNQ1 is preferentially
expressed in the M cells, which is consistent with the lower
IKs density in this region [184]. Transgenic mice overexpressing this spliced variant present abnormalities of SA
and AV function, which suggests a role of KCNQ1 in
normal automaticity [185]. KCNQ1 and KCNE1 expression
and IKs density increases in ventricular myocytes from
hypothyroid rats, but decreases in hyperthyroidism [67]
and in myocytes from infarcted canine ventricle [178].
Mutations in KCNQ1 and KCNE1 are associated with types
1 (LQT1) and 5 (LQT5) of the Romano – Ward variant of the
cLQTS, respectively, and the Jervell– Lange –Nielsen syndrome associated with deafness arises in children who
inherited abnormal KCNQ1 or KCNE1 alleles from both
parents [124,186].
3.2.3.2. Pharmacology of IKs and cellular consequences of
IKs channel blockade. IKs is resistant to methanesulfonanilides, but selectively blocked by chromanols (293B,
HMR-1556) [46,49,187], indapamide [188], thiopentone,
propofol [189] and benzodiazepines (L-768,673, L-735,821
or L-7) [190,191]. The open channel block produced by
chromanols is enantioselective, ()3R,4S-293B and
()3R,4S-HMR 1556 being potent IKs blockers [192,193].
Table 2 summarizes the potency of various drugs to block IKs
and heterologously expressed KCNQ1/KCNE1 channels
[44,130,133,139,190 – 195].
Initially, IKs was considered an attractive target for class
III antiarrhythmic drugs [159]. Because IKs accumulates at
fast driving rates due to its slow deactivation, IKs blockers
may be expected to be more effective in prolonging APD at
fast rates [180]. Indeed, in human ventricular myocytes,
293B prolongs APD and refractoriness in a frequencyindependent manner, so that it is proposed that IKs blockade
might have less proarrhythmic potency as compared to IKr
blockers [46]. Furthermore, because IKs activation occurs at
around 0 mV and this voltage is more positive than the
Purkinje fiber action potential plateau voltage, IKs block
should not be expected to prolong the APD at this level.
Conversely, in ventricular muscle, the plateau voltage is
more positive (c + 20 mV) allowing IKs to be substantially
more activated, so that IKs block would be expected to
increase APD markedly. The net result of both effects might
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3.2.3. The slowly activating component of the delayed
rectifier K+ current, IKs
IKs is slowly activated at potentials positive to 30 mV
with a linear current– voltage relationship, reaching halfmaximum activation at + 20 mV [71,74,179]. Thus, IKs
contributes to human atrial and ventricular repolarization,
particularly during action potentials of long duration and is a
dominant determinant of the physiological heart rate-dependent shortening of APD. As heart rate increases, IKs channels have less time to deactivate, leading to an accumulation
of open channels and a faster rate of repolarization
[152,180]. In guinea pigs, the slow deactivation of IKs
results in a reduction of outward current that contributes
to the slow diastolic depolarization of SA node cells [179].
In guinea pigs, IKs density is greater in atrial than in
ventricular myocytes and in subepicardial and M cells than
in subendocardial cells [118], but is smaller in apical than in
basal myocytes of the rabbit left ventricle [119]. There are
no differences in IKs density among cells from different
regions of the canine right atria [31], while in the canine
ventricle, IKs density is higher in epicardial and endocardial
cells than in the M cells [117] and in right than in left
ventricular M cells [32]. The smaller IKs density in M cells
may explain their steeper APD-rate relations and their
greater tendency to display pronounced action potential
prolongation and to develop early afterdepolarization at
slow heart rates or in response to QT prolonging drugs
[117]. IKs density is downregulated in all layers of the left
ventricle of failing canine hearts [176] and a decrease in IKs
and KCNQ1/KCNE1 mRNA levels is found in myocytes
from infarcted canine ventricle [178].
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J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
3.2.3.3. KCNE1 modifies the pharmacology of IKs. KCNE1
modulates the effects of IKs blockers and agonists. In fact,
KCNQ1/KCNE1 channels have 6 – 100 fold higher affinity
for IKs blockers (293B, HMR-1556, XE991) than KCNQ1
channels [200,201] and the racemate and the enantiomers
of 293B and HMR 1556 have different effects on IKs and
KCNQ1 channels expressed in Xenopus oocytes
[49,193,200]. Mutations in two residues located in the
pore loop and the S5 segment of the KCNQ1 channel
(G272, V307) strongly decrease 293B sensitivity, which
suggests that KCNE1 does not directly take part in
chromanol binding but acts allosterically to facilitate drug
binding to KCNQ1 subunit. Moreover, the IKs activators
mefenamic acid and DIDS have little effect on KCNQ1
channels, although they dramatically enhance KCNQ1/
KCNE1 currents [200] and KCNE1 subunits are also
responsible for the increase in IKs produced by docosahexaenoic, oleic and lauric acids [202].
L-364373 (L3) increases IKs and KCNQ1 currents and
shortens the APD in guinea pig ventricular myocytes [203].
This effect is stereospecific, as S-L3 blocks IKs. Moreover,
R-L3 attenuates the prolongation of the APD and suppresses
the early afterdepolarizations in dofetilide-treated and in
hypertrophied rabbit ventricular myocytes [204]. However,
R-L3 does not affect KCNQ1/KCNE1 channels, which
indicates that KCNE1 interferes with the binding of R-L3
or prevents its action once bound to KCNQ1 subunits [203].
Thus, specific IKs agonists may represent a new strategy to
suppress arrhythmias resulting from excessive AP prolongation in patients with certain forms of LQTS or cardiac
hypertrophy and failure.
3.2.3.4. Structural basis of IKs blockade. In chimeric
constructs of KCNQ1 and KCNQ2 (insensitive to IKs
blockers) expressed in Xenopus oocytes, the molecular
determinants of channel block induced by 293B and L-7
are located in the pore loop (T312) and the S6 domain
(I337, F339, F340 and A344) [191]. Mefenamic acid and
DIDS bind to an extracellular N-terminal segment on
KCNE1 [200] and restore IKs channel gating in otherwise
inactive KCNE1 C-terminal mutants, including the LQT5
mutant D76N [205]. It has been proposed that KCNE1 acts
as an allosteric regulator of channel gating and permeation,
so that in KCNE1 C-terminal mutants the conformational
equilibrium is shifted towards an inactivable closed state
(i.e. a dominant negative phenotype) and DIDS and
mefenamic acid bind to N-terminal residues and shift the
equilibrium towards an activable closed state, leading to
the functional recovery of wild-type IKs phenotype [163].
These results provide interesting clues for the design of
new antiarrhythmic therapies for restoring functional
defects responsible for the LQT5.
3.2.3.5. Regulation of IKs. Lowering [K+]o and [Ca2 + ]o
increase IKs [74,179]. Stimulation of PKA by cAMP,
phosphodiesterase inhibitors and h-adrenergic agonists
increases IKs density and produces a rate-dependent shortening of the APD [206,207]. The KCNQ1/KCNE1 channel
forms a macromolecular signaling complex that is coordinated by the binding of the targeting protein yotiao to a
leucine zipper motif in the C-terminus of KCNQ1. Yotiao
is a scaffold protein which binds and recruits PKA and
protein phosphatase 1 to the channel microdomain and
regulates it via a phosphorylation of a residue (S27)
located in the N-terminal [207]. Disruption of the leucine
zipper domain of KCNQ1 and the mutation S27A prevent
cAMP-dependent up-regulation of IKs, whereas in the
absence of yotiao, KCNQ1/KCNE1 currents are not increased by intracellular cAMP. Phosphorylation of the
KCNQ1 subunit by PKA blunts quinidine- and 293Binduced block, probably by inducing a change in the
KCNQ1 conformation that modifies the drug access to
the blocking site [208]. Thus, h-adrenergic stimulation,
itself a potent proarrhythmic stimulus, may also decrease
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be less drug-induced dispersion in repolarization and reduced risk of arrhythmogenesis [196].
IKs blockers prolong the cardiac APD and QT interval and
suppress electrically induced ventricular tachyarrhythmias in
animals with acute coronary ischemia and exercise superimposed on a healed myocardial infarction [49,159,187].
This QT prolongation can be accentuated by h-adrenergic
stimulation. In arterially perfused canine left ventricle, 293B
prolongs the ventricular APD and the QT interval, but does
not induce TdP. However, in the presence of 293B, isoproterenol abbreviates the APD of epicardial and endocardial,
but not in the M cells, accentuates transmural dispersion
of repolarization and induces TdP [197]. These results
explain the therapeutic actions of h-adrenergic blockers in
patients with LQT1 and LQT5 and the increased risk of
fatal cardiac arrhythmias under physical activity or stressful situations that increase sympathetic activity in these
patients [124,125,198].
Furthermore, under normal conditions 293B and L-7
minimally prolong the APD regardless of pacing frequency
in dog ventricular muscles and Purkinje fibers, probably
because other K+ currents may provide sufficient repolarizing reserve [190]. However, when the repolarizing reserve is
decreased by QT prolonging drugs (IKr or IK1 blockers),
remodeling (heart failure, cardiac hypertrophy) or inherited
disorders, IKs blockade can produce a marked prolongation
of the ventricular APD, an enhanced dispersion of repolarization and TdP arrhythmias [196,199]. Moreover, IKs or IK1
inhibition results in an increased reverse use-dependence in
the presence of an IKr blocker [199]. These findings suggest
that drugs that block several K+ channels are probably more
hazardous than specific channel blockers. Thus, as occurred
with IKr blockers, the characteristics of the IKs blockade
together with the finding of an association between mutations in KCNQ1 and KCNE1 genes and LQT1 and LQT5
[124,125] lowers the initial interest for the IKs blockers as
class III antiarrhytmic drugs.
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
the antiarrhythmic effects of IKs blockers. PKC modulation
of IKs current is complex, suggesting that there are two
functionally distinct PKC sites on KCNQ1 – KCNE1 channels [206]. Moreover, premodulation by PKC prevents IKs
modulation by PKA, and PKC has no effect on IKs current
after potentiation by PKA. These data indicate that IKs is
modulated by PKC and PKA in a mutually exclusive
manner and suggest that multiple interacting phosphorylation sites are involved. Endothelin-1 inhibits the IKs
enhanced by isoproterenol and forskolin and prolongs
APD duration via the ETA receptor pertussis toxin
(PTX)-sensitive G protein/PKA pathway [209].
4. Inward rectifying K+ channels
4.1. The inward rectifier K+ current, IK1
throughout the plateau and open again at potentials negative
to 20 mV. Thus, IK1 contributes to terminal phase 3 of
repolarization. IK1 density is higher in ventricular than in
atrial myocytes, but is similar in epicardial, M and endocardial cells in canine and guinea pig hearts [14]. IK1 is
downregulated in endocardial, epicardial and M cells of
heart failure canine hearts [176]. IK1 density is very low in
SA and AV pacemaker cells and, therefore, the maximum
diastolic potential is more depolarized than in atrial and
ventricular muscle cells [14].
4.1.1. Molecular basis of IK1
Kir2.1 subunits (427 amino acids) encoded by the KCNJ2
gene, coassemble to form tetrameric channels [17]. Several
IK1 channels with conductances of 9, 21, 35 and 41 pS are
recorded in human atrial myocytes [210]. Likewise, different
gene families (Kir2.1 –2.3) have been found in human heart
encoding IK1 [17]. Kir2.1 overexpression increases IK1 density, shortens the APD and hyperpolarizes the resting membrane potential in guinea pig myocytes, while genetic
suppression of IK1 results in opposite changes and in a
pacemaker phenotype accelerated by isoproterenol [211].
The proximal C-terminus and the M2 domain control the
ability of Kir2.1 to interact with other Kir subunits [17].
Table 3
Pharmacological blockade of inward rectifier potassium channels
Drug
Ajmaline
Amiodarone
Azimilide
Bepridil
Bretilium
Cibenzoline
Clofilium
Chloroquine
Diltiazem
Disopyramide
Dronedarone
Flecainide
Glibenclamide
5-Hydroxydecanoate
HMR1098
HMR1556
LY97241
Nicotine
Pentobarbital
Pilsicainide
Pirmenol
Prenylamine
Propafenone
Propranolol
Quinidine
Risperidone
RP58866
Terikalant
Verapamil
IK1
>20 AM
100 AM
IKAch
IKATP
Species
References
145 AM
TXO
GPV,GPA
HAM
TXO
TXO
TXO
TXO
CV
TXO
GPA,TXO
GPA
GPA,GPV
RTV
HEK
HEK
GPV
RTV
CNV
GPV,TXO
GPA
TXO
TXO
RAM, GPA, TXO
TXO
RTV
RVM
GPV,GPA
RTV
TXO
[239]
[213,223]
[42]
[239]
[240]
[240]
[240]
[45]
[239]
[224,239]
[223]
[224,241]
[242]
[243]
[243]
[49]
[47]
[52]
[151]
[224]
[240]
[239]
[54,224,239]
[239]
[12]
[56]
[57,156,225]
[59]
[239]
1 AM
6.3 AM
>2 mM
6.6 AM
3.3 AM
0.3 AM
1.7 AM
c 10 nM
19 AM
135 AM
17.8 AM
17.3 AM
6 AM
81 AM
1.5 AM
>10 AM
1 – 10 AM
4 AM
0.26 – 0.54 mM
25 AM
7.1 AM
0.7 AM
>30 AM
3.4 – 8 AM
>5 AM
2 AM
68 AM
38 AM
63.1 AM
131 AM
67%,10 AM
85 AM
Data are expressed as IC50 values.
CNV: canine ventricular myocytes, CV: cat ventricular myocytes, GPA: guinea pig atrial myocytes, GPV: guinea-pig ventricular myocytes, HAM: human atrial
myocytes, HEK: human embryonic kidney cells, RTV: rat ventricular myocytes, RVM: rabbit ventricular myocytes, TXO: oocytes of X. laevis.
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IK1 is a strong rectifier that passes K+ currents over a
limited range of membrane potentials [17]. At negative
membrane potentials IK1 conductance is much larger than
that of any other current, and so it clamps the resting
membrane potential close to the EK. Upon depolarization,
IK1 channels close almost immediately, remain closed
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J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
4.1.2. Intrinsic gating of IK1
The rectification of IK1 channels results from a voltagedependent blockade of the inner channel pore by cytosolic
Mg2 +, Ca2 + and polyamines (spermine, spermidine, putrescine) [17]. Mg2 + blocks single IK1 channels with a low KD
(10.5 AM at + 30 mV), so that at physiological concentrations of cytoplasmic Mg2 + (f 0.5 –2 mM), the blocking
rate is so fast that inward rectification appears as an
instantaneous process. Polyamine-induced inward rectification depends on two negatively charged residues, D172 and
E224, located in the M2 domain and in the C-terminal
region, respectively [212].
4.1.4. Regulation of IK1
Isoproterenol and forskolin inhibit IK1 in human ventricular myocytes [17,213], suggesting that IK1 can be suppressed by a PKA-mediated phosphorylation of the channel.
Moreover, the effects of isoproterenol are inhibited by
propranolol and acetylcholine [214]. PMA inhibits IK1 in
human atrial myocytes and in Kir2.1b channels but this
effect is inhibited by chelerytrine [215] and mutations of
PKC phosphorylation sites (S64A, T353A). In human atrial
myocytes, methoxamine inhibits IK1 and this effect is
prevented by H-9, a specific PKC inhibitor, suggesting that
a1-adrenergic stimulation reduces IK1 via PKC-dependent
pathways [216]. Kir2.1 channels are negatively controlled
by a tyrosine kinase-dependent phosphorylation process
[217]. Acidosis modulates IK1 but its effect is speciesdependent [17]. PIP2 directly activates IK1, perhaps by an
electrostatic interaction of the negatively charged head
groups of PIP2 with charged residues in the C-terminal
region of the Kir channel [17].
4.1.5. Pathology
IK1 is downregulated in spontaneously hypertensive rats,
in models of cardiac hypertrophy and in patients with severe
heart failure and cardiomyopathies [7,9,35]. The reduction
in ventricular IK1 density is transmurally homogeneous in
failing canine hearts [176]. Reduced IK1 density is observed
in canine Purkinje cells after myocardial infarction [8] and
in a 3-day-old infarcted rat heart, but this reduction is
4.2. The acetylcholine-activated K+ current, IKAch
Acetylcholine activates K+ channels (KAch) in pacemaker SA and atrial cells, thus regulating heart rate [19].
IKAch density is c 6 times greater in the atrium than in
the ventricle [14] and results from a tetrameric complex
of two Kir3.1 (501 amino acids) and two Kir3.4 (419
amino acids) subunits [221]. Following the binding of
acetylcholine to muscarinic M2 receptors, the Ghg subunits of the PTX-sensitive G protein (Go/Gi family)
activate the KAch channel by interacting directly to its
cytoplasmatic N- and C-termini. Activation of IKAch
hyperpolarizes the membrane potential, slows the spontaneous firing rate of the pacemaker cells of the SA and
AV nodes and delays AV conduction [1,12]. These effects
explain why vagal maneuvers or intravenous adenosine
can terminate atrioventricular reentrant tachyarrhythmias.
IKAch activity can be stimulated by intracellular ATP, PIP2
and ETA endothelin, A opioid, a2-adrenergic and A1
adenosine receptor agonists [11,12,222] and inhibited by
intracellular acidification and several antiarrhythmic drugs
(Table 3) [223 – 225]. Disopyramide, procainamide and
pilsicainide mainly block the muscarinic receptors, while
flecainide and propafenone act as open channel blockers
[224]. Atrial KAch channels are inhibited by membrane
stretch, possibly serving as a mechanoelectrical feedback
pathway, a property conferred by the Kir3.4 subunit
[226]. Vagal stimulation produces a nonuniform shortening of the atrial APD and refractoriness mediated by
activation of IKACh, an effect that may contribute to the
perpetuation of AF [227]. Chronic AF reduces IKAch
density possibly to counteract the AF-induced nonuniform
shortening of the atrial refractoriness [219].
4.3. ATP-sensitive K+ channels, KATP
Cardiac KATP (f 70 –90 pS) are inhibited by physiological intracellular ATP levels (ATPi), coupling cell metabolism to membrane potential [18,228]. The mechanism of
inward rectification involves an open channel blockade by
intracellular Mg2 + and Na+ ions [229].
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4.1.3. Pharmacology of IK1
Ba2 + is a potent IK1 blocker (IC50 = 20 AM) [210]. Other
IK1 blockers are summarized in Table 3 [42,45,47,49,
52,54,56,57,59,151,213]. IK1 blockers prolong atrial, AV
node and ventricular APD and are effective against various
types of experimental reentrant ventricular tachycardias
[159]. Moreover, IK1 blockers produce membrane depolarization (an effect that slows conduction velocity due to a
voltage-dependent inactivation of Na+ channels) and prolongs the QT interval, both actions being proarrhythmic.
Fast heart rates increase the [K+] in the narrow intercellular
space to several mM and the IK1 density, which results in a
shortening of the APD that might offset the ability of IKr
blockers to prolong the APD under these conditions.
greater in epicardial than in endocardial myocytes [66].
The ventricular myocytes from patients with idiopathic
dilated cardiomyopathy exhibits decreased channel activity,
longer APD and lower resting membrane potentials than
those from patients with ischemic cardiomyopathy [218].
The downregulation of IK1 produces membrane depolarization, prolongation of the APD and both early and delayed
afterdepolarizations [7,9]. However, IK1 density increases in
patients with chronic AF [219]. Mutations of KCNJ2
encoding Kir2.1 result in dominant negative effects on the
current and have been associated with Andersen’s syndrome, an inherited disease characterized by periodic paralysis, dysmorphic features, QT prolongation and ventricular
arrhythmias [220].
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
4.3.1. Molecular basis of IKATP
Cardiac KATP results from the coassembly of four inwardly rectifying channel a-subunits (Kir6.2) and four
regulatory SUR2A subunits (Fig. 2B) [18,228]. Kir6.2
subunits confer inhibition by ATP [230]. The SUR2A
subunit has three transmembrane domains (TMD0, TMD1,
and TMD2), each of which consists of five, five, and six
membrane spanning regions and two nucleotide binding
folds (NBF-1 and NBF-2) located in the loop between
TMD1 and TMD2 and in the C-terminus, respectively
[18,231]. The SUR2A subunit confers sensitivity to
MgADP, sulfonylureas and K+ channel openers (KCOs)
and ATP hydrolysis at each NBF gates the K+ permeation
through the Kir6.2 a-subunit [18,228]. There is an endoplasmic reticulum retention motif (RKR) in the C-terminal
region in Kir6.2 and in an intracellular loop between TMD1
and NBF-1 in SUR2A that prevents their surface expression
in the absence of the other subunit [232].
4.3.3. Pharmacology of IKATP
KATP are inhibited by ATPi and activated by MgADP, so
that the channel activity is regulated by the ATP/ADP ratio
[16]. Several factors desensitize KATP to inhibition by ATPi,
including nucleotide diphosphates, lactate, oxygen-derived
free radicals and adenosine A1 receptor stimulation
[18,228].
KCOs (pinacidil, cromakalim, rimakalim and nicorandil)
bind at two distinct regions of TMD2, the intracellular loop
joining TM13 and TM14 and TM16 and TM17 (residues
K1249 and T1253) [18,228,236]. They exert cardioprotective effects in experimental models of myocardial ischemia/
reperfusion and in patients with acute myocardial infarction
[159,233]. However, KCOs also activate vascular KATP
(Kir6.1/SUR2B) and produce hypotensive effects that limit
their use in the treatment of myocardial ischemia. Moreover,
since IKATP density is larger in the epicardium, KCOs
produce a more marked shortening of APD in epicardial
cells, leading to a marked dispersion of repolarization and to
the development of extrasystolic activity via a mechanism of
phase 2 reentry [237]. However, KCOs shorten the APD
(QT interval), reduce transmural dispersion of repolarization
and suppress early and late afterdepolarizations induced in
patients with LQT1 [238]. Thus, KCOs may prevent spontaneous TdP when congenital or acquired LQTS is secondary to reduced IKr or IKs.
KATP are blocked by sulfonylureas (i.e. glibenclamide,
glicazide, glipizide, glimepiride, tolbutamide), glinides
(repaglinide, nateglinide) and various antiarrhythmic drugs
(Table 3) [12,239 – 243]. The sulfonylurea binding sites
include the region between TM15 and TM16 in TMD2
and S1237, located in the loop between TM15 and TM16
[18,244]. Cardiac IKATP blockers prevent the shortening of
the APD and the incidence of ventricular fibrillation during
the myocardial ischemia, even when in models of ischemia –
reperfusion they are mainly arrhythmogenic [159,233,234].
The clinical effects of glibenclamide are also contradictory in
type 2 diabetic patients with coronary artery disease [245].
Moreover, since KATP are present in pancreatic h-cells and
smooth muscle [18], IKATP blockers can produce hypoglycemia and coronary vasoconstriction, effects that may preclude their interest as antiarrhythmic agents. Cardioselective
IKATP blockers (clamikalant, HMR 1098) inhibit hypoxiainduced APD shortening and prevent ventricular fibrillation
induced by coronary artery occlusion in post-infarcted conscious dogs at doses that have no effect on insulin release,
blood pressure or coronary blood flow [233,234]. Thus, they
may represent a new therapeutic approach to the treatment of
ventricular arrhythmias in patients with coronary heart
disease.
Recently, it has been postulated that mitochondrial KATP
(mitoKATP) rather than sarcolemmal KATP (sarcKATP) are
responsible for IPC [233]. In fact, diazoxide, a selective
mitoKATP agonist mimicks IPC, whereas 5-hydroxydecanoate (5-HD), a selective mitoKATP blocker, suppresses the
cardioprotection induced by IPC and diazoxide. Since
cardiac mitoKATP appear to be pharmacologically distinct
from sarcKATP, it seems possible to develop selective
mitoKATP openers devoid of hemodynamic and cardiac
electrophysiological side effects of first generation KATP
openers. The selective mitoKATP opener BMS-191095
exerts cardioprotective effects without shortening APD,
which may explain the reduced propensity for reentrant
arrhythmias [246]. However, recent evidence has demonstrated that diazoxide also activates sarcKATP channels
[247], an effect that is inhibited by HMR1098 but not by
5-HD and that it does not improve contractile function in
Kir6.2 / mice [248]. These data suggest that activation
of sarcKATP rather than of mitoKATP is responsible for its
cardioprotective effects. Therefore, further research is needed to determine the molecular basis of mitoKATP and the
cardioprotection induced by specific mitoKATP openers
[233].
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4.3.2. Role of KATP channels
Opening of KATP during myocardial ischemia shortens
the APD and decreases Ca2 + influx through L-type channels. Both effects prevent cardiac Ca2 + overload, preserve
ATP levels and increase cell survival [233,234]. Activation
of KATP plays an important role in ischemic preconditioning
(IPC), wherein single or multiple brief periods of myocardial ischemia confer protection against a subsequent prolonged ischemia, reducing myocardial infarct size, severity
of stunning and incidence of cardiac arrhythmias. However,
activation of KATP also resulted in shortening of the APD,
accumulation of extracellular K+, membrane depolarization
and slow conduction velocity, effects that render the ischemic heart vulnerable to reentrant arrhythmias [159].
Kir6.2 / mice lack KATP and present an aberrant regulation of cardiac excitability, inadequate Ca2 + handling,
ventricular arrhythmias and sudden death following sympathetic stimulation [235]. These results suggest that Kir6.2 is
required for adaptation to stress.
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26
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
5. Two pore or background K+ channels, K2P
Cardiac cells have K2P currents with little time- or
voltage-dependency, termed background currents, that regulate resting membrane potential and cell excitability (Fig.
2C) [20 – 22]. K2P are insensitive to conventional K+ channel blockers (4-AP, TEA, Ba2 +, Cs+, glibenclamide), but
show differential sensitivity to membrane stretch, changes in
pH, fatty acids and inhalation (e.g. halothane, isoflurane,
chloroform) and local (e.g. lidocaine, bupivacaine) anesthetic agents and are regulated by second messenger phos-
Table 4
Pharmacological characteristics of leak channels
Parameter
TASK
TWIK
TREK
THIK
Stretch
Arachidonic acid
Acid internal (pH 6)
Acid external/pH 6)
PKA activators (1)
PKC activators (2)
Halothane
Isoflurane
Chloroform
Lidocaine
Bupivacaine
Ropivacaine
0
0
0
#
0
0
za
z
z
#
#
#
0
0
#
0
0
z
zzz
z
z
0
#
#/0
za
0
z
0
z
#
#
z
0
#
#: currents inhibited, z: currents increased, 0: no effects, a: activates an
inactive channel.
PKA: protein kinase A, PKC: protein kinase C.
(1) PKA was activated with cAMP, forskolin and 1-methyl-3-isobutylxanthine (IBMX).
(2) PKC was activated with phorbol-12-myristate 13-acetate (PMA).
phorylation (Table 4) [22]. Cardiac expression of TWIK-1
may underlie the background K+ current active during the
plateau of the action potential (IKp) [20].
TASK currents are highly sensitive to variations in the
extracellular pH in the physiological range. Mutations of the
residue H98 (H98D) which lies immediately distal to the
first pore domain reduces pH sensitivity [22]. TASK currents are insensitive to arachidonic acid, stretch and PKC
activators, but are inhibited by local anesthetics and submicromolar concentrations of the endocannabinoid anandamide and increased by volatile anesthetics [22]. TREK and
TRAAK channels are activated by membrane stretch, cell
swelling, negative pressure and lysophosphatidylcholine.
TREK and THIK-1 are stimulated by arachidonic acid and
polyunsaturated fatty acids and THIK-1 channels are
inhibited by intracellular acidification. TWIK currents are
increased by arachydonic acid and PKC activation and
inhibited by intracellular acidosis, but are insensitive to
PKA activation. In contrast, TREK channels are modulated
by Gq-, Gi- and Gs-coupled receptors, inhibited by activators
of PKA and PKC and increased by intracellular acidosis.
Thus, far from being passive leaks, as initially thought, K2P
channels are sensitive to changes in pH, membrane stretch
and phosphorylation state, possibly acting as cellular sensors and tranducers, translating these stimuli into a change
in the membrane potential and excitability [22]. If so, some
K2P channels will represent important therapeutic targets in
the near future.
6. Conclusions and perspectives
Cardiac K+ channels have been recognized as potential
therapeutic targets. However, the rational design of safer and
more effective K + channel blockers/openers and the
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4.3.4. Regulation of KATP
Both PKC and adenosine A1 receptor activation enhance
KATP and induce IPC and these effects are suppressed by
chelerythrine or bisindolylmaleimide [249]. The effects of
PKC on KATP activity are associated with phosphorylation
at T180 in the Kir6.2 subunit. KATP activated by glucosefree anoxia close immediately upon reoxygenation, but
activation of both novel and conventional PKC isoforms
contributes to the persistent opening of the channels under
these circumstances [250]. KATP can be activated indirectly
by PKA-coupled and other protein kinase-coupled receptors
via phosphorylation of channel proteins or by depleting
cellular ATP levels [244]. The PKA site responsible for
channel stimulation includes the residues S372 and T224.
PIP2 directly interacts with positively charged residues
(R176, R177) at the C-terminus of Kir6.2 subunit stabilizing
the open state of the channel and antagonizes ATP inhibition
of KATP [17,251], whereas breakdown of PIP2 by PLC
enhances the ATP-sensitivity of KATP channels [252]. Moreover, PIP2 mimics ATP in preventing current rundown and in
rescuing activity after rundown [251]. Protein tyrosine
kinases (PTKs) are also mediators of ischemic preconditioning. In guinea pig ventricular myocytes the PTK inhibitor
genistein elicited IKATP. Stimulation of receptor PTKs with
epidermal growth factor, nerve growth factor or insulin
attenuates, while the protein tyrosine phosphatase inhibitor
orthovanadate prevents the effects of genistein on IKATP
[253]. These results suggest that the PTK-protein tyrosine
phosphatase signaling pathway may be one of the regulators
of cardiac sarcKATP.
The release of nitric oxide (NO) and bradykinin during
ischemia can also play a role in IPC. KATP are activated
via the NO-cGMP-PKG signaling pathway, which phosphorylates some serine – threonine residues and this activation is reversed by protein phosphatase 2A [254]. PKC-q
and PKC-D are activated by NO donors in rabbit hearts
[255] and their infarct-size limiting effects are abolished by
chelerythrine. Interferon-a inhibits IKATP in rabbit ventricular cells and this effect is blocked by genistein, but is not
affected by H-7, an inhibitor of PKC and PKA [256].
These findings suggest that tyrosine kinase-mediated inhibition of IKATP by cytokines may aggravate cell damage
during myocardial ischemia.
J. Tamargo et al. / Cardiovascular Research 62 (2004) 9–33
Other possible targets include accessory h-subunits and
intracellular signalling molecules (PKA, PKC, cyclic
nucleotides). Finally, to optimize drug – channel interaction
thousands of chemical entities must be screened to identify
a few candidate drugs. The new technological developments (planar-array chip-based approaches) address the
high throughput restrictions of classical patch clamp electrophysiology and allow recordings from hundreds of cells
per day and provide new perspectives for drug discovery.
All this information is the basis to understand the pathophysiology and arrhythmogenesis of the diseased hearts
and to identify new targets for the development of K+
channel modulators safer and more effective than those
actually prescribed.
Acknowledgements
This work was supported by Comisio´n Interministerial de
Ciencia y Tecnologı´a (SAF2002-02304), Comunidad Auto´noma de Madrid (08.4/0038.1/2001) and Pfizer Foundation
Grants.
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attempts to prevent the proarrhythmic effects linked to the
blockade of cardiac K+ channels should be based on a better
understanding of the molecular basis of the target channel,
its cardiac distribution and function and the type of drug –
channel interaction. The past decade over 80 human genes
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Ion channel blockers tested in clinical trials have shown
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27
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