Download Regional expression of cardiac ion channels and

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Regional expression of cardiac ion channels and
cardiac electrical activity Gerno t Schra m M.D., Ma rc Po urrier B.Sc., Peter Melnyk B.Sc., Stanle y Nattel M. D.
Introduction
Important differences in electrophysiological properties have been noted
between different regions of the heart. Electrophysiological heterogeneity
has also been detected within different parts of a given tissue, such as
the ventricular subendocardium, midmyocardium and subepicardium.
Although many molecular candidates for native ionic currents have been
identified, the molecular basis of most currents is not completely
understood. Heterogeneity of channel protein composition might well
underlie the differences observed among the properties of ionic currents
or the shape of the action potential in different regions of the heart.
Techniques such as immunocytochemistry, immunohistochemistry and
Western blotting have played an important role in identifying tissue
expression of channel proteins as well as their cellular localization. This
review summarizes the electrophysiological differences observed in
different regions of the heart and correlates them with information
regarding expression patterns of ion channel subunits that might account
for regional functional variation.
The inward rectifier current, I K1
IK1 is responsible for the maintenance of the
cardiac resting potential and the final phase of
cardiac repolar ization. It is believed that
differences between atrial and ventricular IK1 are
responsible for the differences observed
between the atr ial and ventricular action
potential. The atrial action potential has a short
plateau phase and relatively slow repolarization,
whereas the ventricular action potential has a
long plateau and a much faster repolarization
phase.1 Patch clamp studies have revealed
significant differences betw een atr ial and
ventricular IK1. Atrial IK1 has a 4-10 fold lower
current density and conducts less outward
current than ventricular IK1.1-3 It is believed that
a smaller I K1 contributes to the less negative
r esti ng potenti al and s low er ter mi nal
repolarization in atrial cells.2
Furukawa et al reported a lar ger outward
component of I K1 in endocardial cells than in
epicardial cells in the cat. No differences in
si ngl e channel conduc tance and open
probability were observed, suggesting that
observed differences are due to differences in
current density and not to differences in
mol ecul ar c omposi tion. 4 H ow ever, no
differences in IK1 current density or kinetics have
been found across the left ventricular wall in
myocytes isolated from guinea pig5 or dog.6 The
observed discrepancies might be due to
Re la t e d P roduc t s
Anti-Kir2. 1
Anti-Kir2. 3
Cat. # APC-026
50 µl $98.00
0.2 ml $320.00
Cat. # APC-032
50 µl $98.00
0.2 ml $320.00
(IR K1, Kcnj2)
Host: Rabbit
Epitope: Pept ide corresponding to amino acid residues 392-41 0
of hum an Kir2.1.
Epitope location: Intracellular, C-t erminal part.
Homology with other species: R abbit, bovine, pig,
guinea pig - identical; rat , mouse - 17/19 residues identica l;
chic ken -15/19 residu es iden tic al;
pigeon - 14/18 residues identica l.
Reactivity confirmed: R at.
Western blotting: Rat brain mem branes (1:200).
Immunohistochemistry: R at brain sect ions.
Control antigen included in price.
References using this antibody:
1. Keren-R aif man, T. et al. (2000)
Bio chem. Bio phys. Res. Comm un. 274, 852.
(IR K3, BIR 11, Kcnj4)
1
2
3
Western blotting of rat
brain (1,3) or kidney
(2,4) membranes:
1,2. Anti-Kir2.3 antibody
(#APC-032) (1:200)
3,4. Anti-Kir2.3
antibody, preincubated
with the control peptide
antigen.
1
4
97
66
45
31
2
97
66
45
31
Western blotting of rat brain
membranes:
1. Anti-Kir2.1 (#APC-026)
(1:200).
2. Anti-Kir2.1,
preincubated with the control
peptide antigen.
Regional expression of cardiac ion channels and cardiac ele ctrical activity
Host: Rabbit
Epitope: Pe ptid e corresp onding to a mino acid residues
418-437 of rat Kir2.3.
Epitope location: Intracellular, C-t erminal part.
Homology with other species: M ouse - id entical; g uine a pig
- 19/20 residies identical; hamster, human, Xenop us - 18 /20
residues identical.
Reactivity confirmed: R at.
Western blotting: Rat brain me mbranes, rat kidney
membranes (1: 200).
Immunohistochemistry: Rat brain sect ions.
Control antigen included in price.
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M olecula r Tools for the Neuroscie nc e Co mm unity
species-specific variations.
Clones of the Kir2 family are believed to
underlie IK1. Four different alpha subunits have
been cloned and shown to be present in the
human heart.2, 7-9 Kir2.1 mRNA is the most
abundant subunit, with similar concentrations in
atrium and ventricle.2 Kir 2.2 mRNA expression
was relatively weak in both atrium and ventricle.
Kir2.3 concentration is approximately 12-fold
higher in atrium than in ventricle, but about 10fold lower than Kir2.1.2 The abundance of Kir2.4
in human heart has not been determined. It has
been speculated that the comparatively high
abundance of Kir2.3, a lower-conductance
subunit, in atrium might contribute to the smaller
current density of atrial IK1.2 Recent data
suggests that Kir5.1 can act like an endogenous
dominant negative subunit, co-assemble with
Kir2.1 and lead to the formation of nonconducting inward rectifier channels in the
brain.10 It is unclear if this mechanism operates
in the heart. Nothing is known at present about
the cellular localization of Kir2 subunits.
The delayed rectifier current, IK
IK consists of two distinct components, the
rapidly activating I Kr and the slowly activating
IKs .11 IK is the main phase 3 repolarizing current
in the heart. Comparison of IK between canine
right and left atrium showed a consistently
higher current density of IKr in the left atrium.12
No differences in current kinetics were
observed. Western blot experiments showed a
significantly greater expression of human ethera-go-go-related gene (Herg) protein in the left
atrium, consistent with the electrophysiological
data. No significant differences were found in IKs
current densities.12 These results are significant,
given the prominent role of the left atrium in AF
maintenance,13 which may in part be due to
abbreviated left atrial refractoriness due to
larger IKr.
Comparison of Herg protein expression with
functional IKr expression in rat found Herg
protein and IKr to be more prominent in atria
than in ventricles, whereas Herg pr otein
expression in mice and humans was higher
in the ventricle. 14 RNase protection assays
found similar levels of Herg mRNA in rabbit left
atrium, left ventricle, SA node and in canine
Pur kinje fi br es, left and r ight ventr icle
suggesting that either interspecies differences or
post-translational mechanisms may lead to
differences in the level of expressed protein.
Herg mRNA levels are ~1.5-fold more abundant
than those of Kv4.3 in canine right ventricle.15
Voltage clamp studies using left ventricular
guinea pig myocytes revealed a higher IK current
density in sub-epicardial and mid-myocardial
than in endocardial myocytes.
Both components, IKr and I Ks, were significantly
smaller i n sub-endocardial than in midmyocardial or sub-epicardial cells.16 Furukawa
et al. reported a higher current density of IK, with
faster activation and delayed deactivation, in left
ventricular epicardial compared to endocardial
cells of the cat. No changes were found in open
probability and single channel conductance
leading to the suggestion that the observed
differences in current density are due to a higher
expression of channel protein and not to
differences in molecular composition.4
Liu and Antzelevitch described a smaller IK in
mid-myocardial cells compared to cells isolated
from the endocardium or epicardium in dogs.
Further evaluation showed that the observed
difference was due to a significantly smaller IKs
in mid-myocardium. No changes were found in
the current density of IKr nor the kinetics of IKr or
IKs in the three cell types.17 In ferret, Herg mRNA
transcripts and protein levels were found to be
higher in epicardial cell than endocardial cell
layers.18
To further investigate the lower IKs density in
mid-myocardial cells Péréon and colleagues
Re la t e d P rodu c ts
Anti-GIRK1
Anti-TASK-1
(Kir 3.1, Kcn j3)
(TWIK-Relate d Acid Sen sitive K+ C hannel, TASK,
cTBAK-1, Kcnk3)
Cat. # APC-005
50 µl $98.00
0.2 ml $320.00
Host: Rabbit.
Epitope: GST f usion p rotein w it h sequence corresponding
t o re sidues 436-501 o f mouse GIRK1.
Epitope location: Intrace l ular, C -terminal part .
Homology with other species: Rat - iden tic al; hum an,
g uine a pig, chicken - respect iv ely, 64 /66, 63/6 6, and 59/66
resid ues ide ntic al.
Reactivity confirmed: Rat, mouse
Western blotting: Rat brain membranes (1:200).
Immunohistochemistry: Ra t bra in sections.
Control antigen included in price.
References usin g th is antib ody:
1. Kuzhikandathil, E.V. et al. (1998) Mol. Cell. Neur osci. 12, 390.
2. Kennedy, M.E. et al. (1999) J. Biol. Chem. 274, 2571.
3. Pei, Q . et al. (1999) Neur oscience 90, 621.
4. Singer- Lahat, D. et al. (2000) Eur J. Physiol. 440, 627.
5. Lei, Q. et al. (2000) Pr oc. Natl. Acad. Sci. USA 97, 9771.
6. Jelacic, T.M. et al. (2000) J. Biol. Chem. 275, 36211.
1
Western blotting of rat brain
membranes:
1. Anti-TASK-1 (#APC-024)
(1:200).
2. Anti-TASK-1,
preincubated with the control
peptide antigen.
1
2
250
148
60
42
30
22
2
97
66
45
37
Western blotting of rat brain
membranes:
1. Anti-GIRK1 (#APC-005)
(1:200).
2. Anti-GIRK1,
preincubated with the control
antigen.
Regional e xpression of cardiac ion channels and cardiac electrical activity
Cat. # APC-024
50 µl $98.00
0.2 ml $320.00
Host: Rabbit
Epitope: Pept ide (C)EDEK RD AEH RALLT R NGQ,
correspond ing to amino a cid residu es 252-269 of hu man
TASK-1.
Epitope location: Intracell ular, C-terminal part.
Homology with o ther species: rat, mouse - 17/1 8 residues
ide ntic al.
Homology with human TASK-3: 11/ 17 residues identical.
Reactivity Confirmed: R at.
Western blotting: Rat brain membranes (1:200).
Immunohistochemistry: R at brain se ctions (see also 3)
References using this an tibod y:
1. Lopes, C.M.B. et al. ( 2000) J. Biol. Chem. 275, 16969.
2. Millar, J.A. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 3614.
3. Kindler, C .H. et al. ( 2000) Brain Res. Mol. Br ain. Res. 80, 99.
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M olec ul ar Tool s f or the Ne uro sc ience C om m unit y
used RNase protection assays to determine
KvLQT1 message expression across the human
right and left ventricular wall. Overall KvLQT1
express ion w as simil ar throughout the
ventricular wall. Midmyocardial cells, however,
were found to contain a higher percentage of an
endogenous N-terminal truncated KvLQT1
splice variant, referred to as KvLQT1 isoform 2,
which acts as a dominant negative.
Co-expression of isoform 1 with isoform 2 in
COS 7 cells in a stoichiometry mimicking that in
midmyocardium resulted in a 75% reduction in
current amplitude, consistent with the data
provided by Liu & Antzelevitch. Current kinetics
were not changed by co-expression with
KvLQT1 isoform 2. KvLQT1 concentrations in
atrium were found to be similar to those in
endocardial and epicardial left and ri ght
ventricular cells. The regulatory subunit minK
w as found to be simi lar ly expressed in
midmyocardium, endocardial and epicardial left
and right ventricular cells.
The transient outward K + current, I to
Regional differences in Ito are well described
and contribute to the observed differences
in action potential configuration in various
species.19-23 Ito density has been found to be
greater in epicardial cells than in cells isolated
from the endocardium in both dog 20 and rat.21,22
In human ventricle, Ito is larger in sub-epicardial
than in subendocardial myocytes.24 Differences
in inactivation kinetics and 4-AP sensitivity have
been found between atrial and ventricular
human my oc ytes sugges ting pot enti al
differences in their molecular basis.23 Action
potentials recorded from regions with a high I to
current density show a typical “spike and dome”
configuration.20
Based upon differences in activation and
inactivation kinetics, as well as recovery from
inactivation, Ito1 has been divided into two types,
fast (It o,f) and slow (I to,s ).2 5-31 Ito, s density is
high in left ventricular epicardial myocytes in
humans 28 and ferret.27 It is believed that Kv4.2
and Kv4.3 contribute to the rapid component Ito,f
and v1.4 appears to underl ie the sl ow
component Ito,s.22 Important differences among
various species, as well as differences in the
distribution of these subunits across the
ventricular wall, are well documented. The
distribution of various subunits contributing to Ito
has been most extensively studied in rat. In the
atrium, only Kv4.2 has been found,32 whereas
both Kv4.2 and Kv4.3 have been identified in
ventricle33 Kv4.3 and Kv1.4 mRNA expression
across the ventricular wall of rat heart is almost
uniform, whereas Kv4.2 show s a marked
gradient with higher expression levels in
epicardial than endocardial cells.34, 35 Kv4.2
expression has been shown to parallel I to
current density across the left ventricular wall34
suggesting that Kv4.2 is the main subunit
underlying Ito in rat.
Kv4.2 express ion pr edominates in the
right ventricular wall.22 Confocal and
immunohistochemical studies show that Kv4.2 is
localized in the transverse-axial tubular system
of the rat myocytes.36 Kv4.3 and Kv1.4 may be
proportionately more important in the septum.22
Important differences have been found among
various species. In the mouse, Kv1.4 has been
found in high concentration in the septum and is
believed to underlie Ito,s, whereas alpha subunits
of the Kv4 subfamily underlie I to ,f in the
ventricular apex and septum.37 The properties of
It o in rabbit myocytes are similar to those of
Kv1.4, suggesting an important role of Kv1.4 in
r abbit It o . 38 - 40 In situ hybr idization and
immunohistochemistry has shown regional
differences in the expression of Kv1.4 and
Kv4.2/4.3 in ferret heart, suggesting that Kv1.4
and Kv4.2/4.3 underlie Ito in ferret left ventricular
endocar di al and epic ardi al myoc ytes ,
respectively.27 The molecular basis for canine
and human It o has been attributed to Kv4.3.35
RNase protection assays showed that neither
Re la t e d Pr oduc t s
Anti-Kv1.5
A
B
C
(Kcn a5)
SON
Cat. # APC-004
50 µl $98.00
0.2 ml $320.00
Host: R abbit
Epitope: GST fusion protein w ith seq uence corresp ondin g to
residue s 513-602 of mou se Kv1.5.
Epitope location: Intra cellular, C-termin us.
Homology with other species: Rat, rabb it, human, bovine,
dog - respectively, 86/90, 71/90, 70 /90, 66/ 90, and 66/90
residue s ident ic al.
Reactivity confirmed: Rat, mouse, horse .
Western blotting: R at brain membranes (1: 200).
Immunohistochemistry: Rat brain sections (see als o 9)
Immunoprecipitation.5 ,7
Control antigen included in price.
1
2
250
148
Immunohistochemical staining of Kv1.5 using Anti-Kv1.5 (#APC-004) in the
paraventricular nucleus (PVN) and supra-optic nucleus (SON) in (A). In the PVN (B),
higher power magnification shows staining of some neurons (triangle) and of axons
which had varicosities (arrows). In the supra-optic nucleus (C), higher power
magnification shows staining of cells (triangles) and axons with varicosities
(arrows). All patterns were eliminated by pre-incubation with Kv1.5 control antigen.
60
42
30
Western blotting of rat brain membranes:
1. Anti-Kv1.5 (#APC-004) (1:200).
2. Anti-Kv1.5, preincubated with the
control antigen.
R egional expre ssion of ca rdiac ion channels and cardiac electrical activity
R ef eren ces using t his ant ibod y:
1. Hu, X.Q. et al. ( 1998) J. Biol. C hem. , 273, 5337.
2. G uo, W . et al.. (1997) Am. J. Physiol. 272, H2599.
3. G uo, W .et al.( 1997) Eur. J. Physiol. 434, 206.
4. Clement- Chomienne, O. et al.(1999) J. Physiol. 515.3, 653.
5. Sobko, A. et al. ( 1998) J. Neurosci. 18, 10398.
6. Sobko, A et al. (1998) EMBO J. 17, 4723.
7. Maruoka, N.D. et al. (2000) FEBS Letter s 473, 188.
8. Yamashita, T. et al. ( 2000) Circulation 101, 2007.
9. Chung, Y.H. et al. (2000) Brain Res. 875, 164.
10. Peretz, A. et al. (2000) T he EMBO J. 19, 4036.
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Kv4.1 nor Kv4.2 mR NA is expressed at
detectable levels in canine ventricular muscle,
whereas Kv4.3 is abundant. Kv4.3 mRNA is
expressed in human ventricle at similar levels to
those found in canine ventricle.35
Han et al. reported the presence of TEAsensitive Ito in canine Purkinje fibers. Their data
suggest that the molecular basis of canine
Purkinje Ito is different from Kv4.2/4.3 and Kv1.4,
since these channels are TEA-insensitive. It has
been proposed that Kv3.3 and Kv3.4 subunits
might play an important role in Purkinje Ito, but
the exact molecular basis still remains to be
determined.41
The Na +-Ca 2+ exchanger (NCX)
The Na+-Ca2+ exchanger (NCX) catalyzes the
exchange of three Na+ for one Ca2+ across the
plasma membrane in many mammalian cells.42
The transport is reversible and can facilitate
Ca2+ entry, leading to Ca2+ release from the
sarcoplasmic reticulum.43 The exchange activity
is especially high in cardiomyocytes and plays a
key role in the mai ntenance of C a 2+
homeostasis and relaxation of Ca2+ muscle.42
The exchange of Ca2+ for sodium was first
observed in guinea pig atria.44 Western blotting
and Northern blot experiments have shown
increased expression levels of the NCX1
isoform in heart failure.45-48
NCX proteins in mammals are encoded by three
genes: NCX1, NCX2 and NCX3.49- 51 NCX2 and
NCX3 are found only in the skeletal muscle and
in the br ain.5 2 NCX1 transcripts undergo
al ternative splici ng of 6 i nternal exons
(A,B,C,D,E and F) to produce tissue-specific
isoforms. 53 This splicing confer s distinct
functional characteristics to tissue-specific
isoforms of the Na+ -Ca2+ exchanger.53-56 For
example, the cardiac isoform (NCX1.1) is less
sensi tive to depolarizing voltages and to
activation by [Ca2+ ] i than the renal isoform.
In addition, NCX1.1 is more sensitive to PKA
activation than the renal form.57 Komuro et al
cloned and characterized the human cardiac
Na+ -Ca2+ exchanger in 1992.58
Confocal microscopy of adult guinea pig and rat
heart cells has shown that the NCX is present in
all membranes of the myocytes that face the
extracellular space.59 Dilly et al. showed NCX to
be present on the surface and in T-tubule
membranes on all cardiac myocytes. 60
Immunohistochemistry has revealed a specific
localization in some types of cells.59 Confocal
microscopy has show n that in guinea pig
myocytes, the NCX is located at the intercalated
disks, the transverse tubules and exterior
surface of the membrane.61 In rabbit myocytes,
the NCX appears to be more prominent in
T-tubule membranes than in peripher al
sarcolemma.62
contraction coupling in heart,63- 65 and for the
plateau phase of the action potential.66 L-type
Ca2+ channels cluster in the surface plasma
membrane overlying junctional sarcoplasmic
reticulum in guinea pig myocytes. 67 N, P, Q and
R-type channels have also been identified66,68,69
as high voltage activated calcium channels, 70
but appear not to be expr es sed in
cardiomyocytes.
T-type (”transient”) ICa inactivates very rapidly
The calcium current, ICa
Tw o principal Ca 2+ currents hav e been
described. The voltage- gated L-type (”long
lasting” ) IC a is responsi ble for triggering
sarcopl asmi c reticulum Ca 2+ r elease and
consequently the initiati on of excitation-
Re la t e d Pr oduc t s
Anti-Erg1
Anti-HE RG
(Et her-a-g o-go-re lat ed K+ Channe l 1, Kcnh2)
(Ether-a-go-go-related K + Chan nel 1, Kcnh2)
Cat. # APC-016
50 µl $98.00
0.2 ml $320.00
Cat. # APC-062
Host: Rabbit.
Epitope: Peptide corresponding to residues 1121-113 7 of rat
erg1.
Epitope location: Intracellular, near the C-terminus.
Homology with other species: Mouse - identic al; human, dog,
rabbit - 14/16 residues identic al.
Reactivity confirmed: Rat, huma n, mouse, 2 horse,3 opossum.1
Western blotting: HERG-expressing HEK 293 cells (1:400).
Rat brain membranes, Rat heart membranes (1:200).
Immunohistochemistry: Rat brain sections (see also 2).
Immunoprecipitation.3
Control antigen included in price.
References usin g th is antib ody:
1. Akbarali, H .I. et al. ( 1999) Am. J. Physiol. 277, C1284.
2. Pond, A.L. et al. (2000) J. Biological Chem. 275, 5997.
3. Dr. Lisa C. F reeman, Kansas State Univer sity, personal communication.
1
2
Western blotting of HERGexpressing HEK 293 cells:
1. Anti-HERG (#APC-062)
(1:400).
2. Anti-HERG,
preincubated with the control
antigen.
1
50 µl $98.00
0.2 ml $320.00
205
117
2
205
117 Western blotting of HERG96 expressing HEK 293 cells:
66
45
1. Anti-Erg1 (#APC-016)
(1:200).
2. Anti-Erg1,
preincubated with the control
peptide antigen.
Regional e xpression of cardiac ion channels and cardiac electrical activity
Host: R abbit.
Epitope: GST fusion protein with seq uence correspo ndin g to
residues 1106-11 59 of h uman erg1 (HERG).
Epitope location: Intra cellula r, near the C-t erminus.
Homology with other species: Rabbit - identical; dog, mouse,
rat - re spectively , 51/54 , 50/54, and 50/ 54 residues identical.
Reactivity confirmed: Human.
Western blotting: H ER G-expressing HEK cells (1:400).
Control antigen included in price.
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M olecula r Tools for the Neuroscie nc e Co mm unity
and at relatively negative potentials.64 It is found
mainly in sinoatrial node, Purkinje or atrial cells,
but its expression is also species dependent.64
The fact that I Ca.T is at high density in nodal
cells7 1 and embryonic car diomyocytes 67 ,7 2
suggests that it is important in pacemaker
function.65,67 T-type current is present in the
guinea pig heart but has not been found in rat or
in human adult myocytes.67,73
L-type channels were initially purified from
skeletal muscle.74 They are heteromultimers of
at least 3 different subunits: α1C, β and α 2δ.
The α1C subunit encodes the basic pore-forming
protein of the channel, whereas the auxiliary β
subunit modulates the expression, the open
probability, activation and inactivation.67,75-77 The
α2δ subunit is a disulfide-linked dimer78-80 and is
ubiquitously expressed in all types of high
voltage-dependent Ca2+ channels.81 The α1C
subunit is encoded by three different genes
(CaCh1-3). Only the product of CaCh2a is
present in the heart. Four β subunit genes have
been identified (β1 to β4). Genes encoding ICa.T
were identified in human heart in 1998. 70,82
The molecular basis for T-type calcium channels
has been associated with α1G , α1H and α1I
genes.70,82,83 α1G and α1H are expressed in heart
and brain at different levels.70 The cell or tissue
s pecifici ty has n’t yet been establis hed.
Immunoconfocal and immunogold electron
microscopy labelling were used81 to show the
distribution of L-type Ca2+ channels in cardiac
myocytes isolated from rabbit and rat ventricle.
The channels were localized on the surface of
the plasma membrane and transverse T-tubules
in rabbit myocytes, whereas the labelling was
more intense in T-tubules than in the surface
sarcolemma in rat myocytes.
multiple gap junction phenotypes. These
differ ent phenotypes appear to have a
deliberate and functionally important distribution
patter n thr oughout the hear t. Electron
microscopy has shown SA nodal myocytes to
have small, scattered gap junctions.87 This
finding has implications regarding conduction
velocity in this region, which is very slow.88 The
number and distribution of gap junctions, as well
as their composition, are important factors in
modulating conduction propagation velocity. In a
study of mice heterozygous for a null mutation
in the Cx43 gene (Cx43 +/- mice), it was
determined that this mutation was responsible
for a reduction in the total number of gap
junctions while the remaining gap junctions were
unaffected in terms of size.89 These mice have a
~40% slower ventricular epicardial conduction
velocity.90
Cx43 is the most abundant gap junctional
channel in human91 and rat89 ventricles and
atria. The expression level of Cx43 in the four
chambers of the human heart is more or less
uniform.91 Studies have shown that the human
SA node has little 92 or no Cx43.86 Western
blotting of the AV node reveals a weak signal
found primarily in the middle of the region,86
consistent with the important slowly-conducting
properties of the AV node. When Purkinje fibres
are probed with an antibody for Cx43, the signal
is more intense at longitudinal ends than
transversely.86 This distribution pattern is likely
Connexins/Gap Junctions
Gap junctions are more than simple conduits
allowing for enhanced current flow between
adjacent cardiac myocytes: they allow for the
passage of various cations, anions and small,
non-charged molecules from one cell to another.
Though gap junctions are not directly affected
by membrane potential, they can respond to
differences in transcellular voltage by altering
their conductance.84
As determined by immunocytochemistry, among
the channel proteins expressed in cardiac gap
junctional plaques are three principal connexins:
connexin (Cx)40, Cx43 and Cx45.85 Two other
connexins - Cx37 and Cx46 - have also been
identified in the heart, albeit in trace amounts.86
Different ratios of these connexins give rise to
Anti-Kv4.3
Anti-Kv1.4
Cat. # APC-017
50 µl $98.00
0.2 ml $320.00
Cat. # APC-007
50 µl $98.00
0.2 ml $320.00
(Kcnd3)
Re l at e d Prod uc t s
(Kcn a4)
1
2
96
Host: Rabbit.
Epitope: Peptide corresponding to residues 451 -467 of
h uman Kv4.3.
Epitope location: Intracellular, C -terminal part .
Homology with other species: Rat , ra bbit - iden tic al; mouse
- 16/ 17 re sidues identical.
Reactivity confirmed: Rat.
Western blotting: R at brain membranes (1:200).
Immunohistochemistry: Rat bra in sections.
Control antigen included in price.
References using th is an tibo dy:
1. Yang, E.K. et al. ( 2001) J. Biol. C hem. 276, 4839.
2. Zhang, T.T. et al. (2001) Cir c. Res. 88, 476.
Western blotting of rat
brain membranes:
1. Anti-Kv1.4 (#APC-007)
(1:200).
2. Anti-Kv1.4,
preincubated with the
control antigen.
1
66
45
31
Host: Ra bbit .
Epitope: GST fusion protein with seque nce corre spond ing to
residues 589 -655 of rat Kv1.4.
Epitope location: I ntracellular, C-terminus.
Homology with other species: Mouse - id entical; h uman - 66/67
(or 6 5/67) residues identical, bovine - 64 /67 residues identical.
Reactivity confirmed: Rat, m ouse.
Western blotting: Rat bra in membra nes (1:20 0).
Immunohistochemistry: Rat b rain sections (see also 7-9)
Immunoprecipitation.6
Control antigen included in price.
2
209
125
70
45
32
Western blotting of rat
brain membranes:
1. Anti-Kv4.3 (#APC-017)
(1:200).
2. Anti-Kv4.3,
preincubated with the
control peptide antigen.
Regional e xpression of cardiac ion channels and cardiac electrical activity
References usin g th is antib ody:
1. Attali, B. et al. ( 1997) J. Neur osci. 17, 8234.
2. G uo, W . et al. (1997) Eur. J. Physiol. 434, 206.
3. Yuan, X.J. et al. ( 1998) Am. J. Physiol. 274, L621.
4. Meir i, N . et al. ( 1998) Proc. Natl. Acad. Sci. USA 95, 15037.
5. G uo, W . et al. (1998) J. Mol. Cell Cardiol. 30, 1449.
6. Sobko, A. et al. ( 1998) J. Neurosci. 18, 10398.
7. Mienville, J.M. et al. (1999) J. Neur ophysiol. 82, 1303.
8. Chung, Y.H. et al. (2000) Brain Res. 875, 164.
9. G opel, S.O. et al. (2000) J. Physiol. 528.3, 497.
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I ss ue N o. 15
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M olecula r Tools for the Neuroscie nc e Co mm unity
important in the known functionally-important
anisotropy of conduction.
Cx40 is expressed in human atrium 91 and to a
mu ch l es s er deg r ee i n ve nt r ic ul a r
subendocardium.86 A study of Cx40 expression
in dog also showed that there was a much
higher level of this channel in the crista
ter minalis compared with left ventricular
subepicardium.93 In both man 86 and dog, 85
Purkinje fibres show a far more robust signal for
Cx40 than ventricular myocardium. Canine92
and human86 SA nodal cells clearly express
Cx40. Human AV nodal cells also express
Cx40.86
Low levels of Cx45 can be detected in human
ventricles, with more being seen in the atria.91
Both rat94 and human 86 conduction systems
(including the SA node, AV node and Purkinje
fibres) have an abundant expression of Cx45. In
a recent study of Cx45 knockout mice, it was
found that these mice die of heart failure at
embryonic day 10. Though the hearts of these
mice do contract, conduction block through the
AV node region is observed.95
Four members (HCN1-4) have been isolated
from mouse, rabbit and human tissues,98- 101 and
share a homology of ~60% on the amino acid
level.101 Northern blot showed that only HCN2
and HCN4 are expressed in the human heart.
HCN2 currents activate faster than HCN4. Two
different native currents with distinct kinetics
have been identified in the heart.102-104 Semiquantitative RT-PCR showed that human HCN2
and HCN4 mRNA is found to be approximately
equally abundant throughout the atria and
ventricles. 105 Northern blotting revealed that in
rabbit, HCN4 mRNA is more abundant in SA
nodal cells than in ventricle or atrium. 100 SA
nodal total HCN message in general is 140
times the HCN message for the ventricle and 25
times that of Purkinje fibres,106 corresponding to
the primary pacemaking role of the SA node and
the greater pacemaking activity in Purkinje fibres
compared to working muscle.
distinct β-subunits involved in the modulation of
channel gating and cell surface expression. Ten
genes encoding Na+ channel α-subunits have
been described (NaV 1.1 to NaV 1.9, and NaX )108
and 3 β-subunits ( β1 to β3) have been
identified to date.108-110 Most of the subunits in
the Na V 1.x family have been studied in
heterologous expression systems and,
therefore, the most detailed observations are for
this family. At least 2 α- subunit mRNAs are
expr essed in human heart: NaV 1.5 and
NaV2.1.108,111,112 β1- Subunit mRNA is expressed
in rat and human heart 113,114 but not found
in mouse heart.115 β1 and β2 subunits have
been localized in rat and mouse heart by
immunofluorescence. 111 Both proteins are
The pacemaker current If
A slow membrane depolarization phase between
action potentials is responsible for the rhythmic
activity of the heart.96,97 The principal current
underlying this phenomenon is referred to as If
or Ih. Both Na+ and K + ions carry this current
with the selectivity being fourfold higher for K +.
If is activated on membrane hyperpolarization.
The hyperpolarization-activated cyclic nucleotide
gated cation (HCN) family of channels have
many of the characteristics of If channels.
The sodium current, I Na
INa is responsible for the rising phase (phase 0
upstroke) of action potentials in electrically
excitable cells78, 107,108 and for rapid impulse
conduction through cardiac tissue.107
The functional channel consists of a principal
α pore-forming subunit composed of four
ho mol o gou s d oma i ns ( I- I V) o f s i x
transmembrane segments S1- S6 107 and 2
Re la t e d Pr oduc t s
Anti-Kv4.2
(Sha l1, RK5, Kcnd2 )
Cat. # APC-023
50 µl $98.00
0.2 ml $320.00
1
Host: Rabbit.
Epitope: Peptide corresponding to amino a cid residue s
4 54-469 of rat Kv4.2.
Epitope location: Intrace l ular, C -terminal part .
Homology with other species: Mouse,
h uman - 15/16 residues identical.
Reactivity confirmed: Rat.
Western blotting: Rat brain membranes (1:200).
Immunohistochemistry: Ra t bra in sections.
Immunoprecipitation.3
Control antigen included in price.
2
97
66
45
32
Immunohistochemical staining of rat hippocampus using Anti-Kv4.2 (#APC-023),
counterstained with cresyl violet.
20
Western blotting of rat brain membranes:
1. Anti-Kv4.2 (#APC-023) (1:200).
2. Anti-Kv4.2, preincubated with the control
peptide antigen.
Regional e xpression of cardiac ion channels and cardiac electrical activity
References usin g th is antib ody:
1. Ander son, A.E. et al. (2000) J. Biol. Chem. 275, 5337.
2. Yamashita, T. et al. ( 2000) Circulation 101, 2007.
3. Adams, J.P. et al. ( 2000) J. Neur ochem. 75, 2277.
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M olec ul ar Tool s f or the Ne uro sc ience C om m unit y
expressed in car diac muscle along the Z
lines. 111 The differences in the functional
properties of Na+ channel isoforms result in
unique conductances in specific cell types.108
Immunocytochemical studies have shown that
β1, β2, NaV 1.1 and NaV 1.5 localize along Z
lines in adult rat and mouse cardiac
myocytes. 111,116 NaV 1.5 has been localized to
the surface and T-tubular system of rat hearts
and does not display a large variation other than
a somew hat enhanced labell ing at the
intercalated disks of ventricular myocytes.116
It is possible that this enhanced localization is
related to fast conduction in ventricular
myocardium. In the compact rabbit AV node,
a gradient of Na+ channel expression exists,
with peripherally-located cells having stronger
signals compared with centrally located ones.117
This gradient is likely important in the slow
conduction through this region.
bl o tt i ng , i m mun oc y t oc he mi s t r y an d
immunohistochemistry may help to determine
Kir2 protein expression levels, tissue expression
pattern and cellular localization of Kir2 subunits
giving further insight into the molecular basis of
IK1 . Transmural gradients in I t o are likely
important in governing action potential shape
and expl ai ning many impor tant ECG
phenomena.6,118,120-123 M-cells have longer action
potentials than in other ventricular regions,
playing an important role in arrhythmias due to
excessive prolongation of repolarization (long
QT syndrome). Transmural gradients in IKs, 17
likely related to differential distribution of
KvLQT1 isoforms,124 is probably responsible.
It o dow nr egulati on may be important in
ventricular arrhythmogenesis in heart failure,
and appears to parallel a decrease in Kv4.3
mRNA,125 although the nature of any regional or
transmural differences i s not well-known.
Changes in connexin expression may also
contribute to arrhythmogenesis. For example,
Peters et al. observed a reduction in ventricular
Cx43 signal from hypertrophied and infarcted
human hearts.126
The above are only limited examples of the
importance of regional variations in ion channel
expression in physiological and pathological
states. In addition to differential expression of
por e- for ming subunits, the for mation of
functionally distinct heterotetramers and the
presence of regulatory subunits likely contribute
importantly to regional diversity in channel
function, and relatively little is known about
them. Much more work clearly remains to be
done in this important area.
Functional importance
The cardiac action potential consists of a fine
interplay between a large number of inward and
outward currents of which the main ones are
carried by K+ , Na+ or Ca2+ ions. Characteristic
action potential shapes have been found in
different regions of the heart as well as in
different layers of the ventricular wall. 26,118
Clones encoding a large number of channel
proteins have been identified, some of which
cl e ar l y r ec on st i tut e nat i ve c ha nne l
properti es. 3 5, 3 4 , 94 , 11 9 , 12 0 H owev er, many
discrepancies between native currents and
putative underlying clones remain to be
resolved.
Important regional differences in IK11 are not
readily explained by the mRNA distribution of
corresponding Kir2 family subunits.2
Future studies using techniques like Western
R e f e r e n c e s
1. Giles, W.R. and Imaizumi Y. (1988) J. Physiol. 405, 123.
2. Wang, Z. et al. (1998) Circulation. 98, 2422.
3. Varro, A. et al. (1993) Acta Physiol Scand. 149, 133.
4. Furukawa, T. et al. (1992) Circ Res. 70, 91.
5. Main, M.C. et al. (1998) Exp. Physiol. 83, 747.
6. Liu, D. W. et al. (1993) Circ. Res. 72, 671.
Rel a t ed Pro duc t s
Anti- α 1C
(Ca v1. 2, L -type o f Vo lta ge-Gated
Ca2+ Chan nel, Cacna1 c)
The α1C subunit of voltage-gated Ca2+ channels in
Purkinje cells of the rat cerebellum were visualized
with the Anti-α1C antibody (#ACC-003).
Cat. # ACC-003
50 µl $98.00
0.2 ml $320.00
Host: Rabbit.
Epitope: Pept ide corre spond ing to residues 8 18-835
of rat α1C.
Epitope location: Intracell ular loo p betwe en II and III
domains.
Homology with other species: Mouse - identical; guinea pig
(17/18 residues identical) ; huma n, rabbit (16 /18 residues
iden tic al).
Reactivity confirmed: Rat , mouse, rabbit,2 human.1 0,12
Western blotting: Rat brain membranes (1:200).
Immunohistochemistry: R at brain se ctions
(see also 3,5,7 ,10).
Immunoprecipitation. 2,4
Control antigen included in price.
1
Pictur e contributed
by W. Här tig and J.
Grosche, Leipzig
University, IZKF;
picture obtained
by confocal
laser scanning
micr ospcopy with
Z eiss LSM 510.
2
205
116
Western blotting of rat brain membranes:
1. Anti-α1C (#ACC-003) (1:200).
2. Anti-α1C, preincubated with the control
peptide antigen.
R egional expre ssion of ca rdiac ion channels and cardiac electrical activity
References using this an tibo dy:
1. Bae, I.H. et al. ( 1999) Kor ean J. Biol. Sci. 3, 53.
2. Hu, X.Q . et al. (1998) J. Biol. Chem. 273, 5337.
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5. Lopez, I. et al. (1999) Neur oscience 92, 773.
6. Br undel, B.J.J.M. et al. ( 1999) Cardiovasc. R es. 42, 443.
7. Jiang, Z. et al. (1999) Eur. J. Neurosci. 11, 3481.
8. Serr ano, C.J. et al. ( 1999) FEBS Lett. 462, 171.
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10. Kr euzberg, U. et al. ( 2000) Am J. Physiol. 278, H723.
11. Liu, R. et al. (2000) J. Biol. Chem. 275, 8711.
12. Allard, B. et al. (2000) J. Biol. Chem. 275. 25556.
MO DULATO R
Is sue No. 15
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M O D U L A T O R
alomone labs
M olecula r Tools for the Neuroscie nc e Co mm unity
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125. Kaab, S. et al. (1998) Circulation 98, 1383.
126. Peters, N.S. et al. (1993) Circulation 88, 864.
Re la t e d P roduc t s
Anti-Cardiac α 1 C
(Ca v1 .2a, Cardia c splice form of L-type Voltage -Gated Ca2+
Channe l, Cacna1c)
Cat. # ACC-013
50 µl $98.00
0.2 ml $320.00
1
2
207
Host: Rabbit.
Epitope: GST fusion prote in with sequence corresponding to
residues 1-4 6 of rabbit C av1.2 a,
with serine 44 replaced with alanine.
Epitope location: Intracell ular, N-te rminus.
Homology with other species: R at,
guinea pig - 31/46 residues identica l.
Reactivity Confirmed: R abbit, rat.
Western blotting: Rat h eart membra nes (1:20 0).
Immunohistochemistry.2
Immunoprecipitation.1
Control antigen included in price.
Western blotting of rat ventricular
membranes:
1. Anti-cardiac α1C (#ACC-013) (1:200).
2. Anti-cardiac α1C, preincubated with the
control antigen.
Ref erences u sin g t his antib ody:
1. Shistik E. et al. (1999) J. Biol. Chem. 274, 31145.
2. W ang, X.T., et al. (2000) Am. J. Pathol. 157, 1549.
Regional e xpression of cardiac ion channels and cardiac electrical activity
Address for Correspondence:
Research Center, Montreal Heart Institute
5000 Belanger Str eet East
Montr eal, Quebec, Canada H1T 1C8
S. N attel, M.D.
Dir ector of the Montreal Heart Institute Research Centre
Pr ofessor of Medicine, University of Montreal
+514- 376- 3330 ( telephone)
+514- 376- 1355 ( fax)
email: [email protected] eal.ca
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