Download Interactions of KCNE Auxiliary Subunits with K and other Channels

Interactions of KCNE Auxiliary Subunits with
Kv and other Channels
Alon Meir, Ph.D.
KCNEs are integral membrane proteins that associate with and strongly modulate the
activity of several ion channels. The different KCNE isoforms are widely and differentially
expressed in muscular and neuronal tissues as well as in epithelial cells. Mutations
in KCNE genes were shown to lead to disruptions of diverse physiological systems
and diseases such as cardiac arrhythmias, deafness and paralysis. It is becoming
increasingly apparent that KCNE auxiliary subunits are capable of interacting with many
different ion channels. These interactions are the scope of this brief review.
Functional voltage dependent K+ channels are
constructed primarily by tetramerization of Kv α
subunit pore forming proteins (Figure 1). With
about 40 genes encoding Kv channel subunits,
which may ensemble by either homo or heterotetramerization, the molecular and functional
diversity of native voltage dependent K+ currents
is huge.1
In addition, several groups of proteins referred
to as auxiliary subunits were shown to interact
and modify Kv currents. These include Kvβ, KChiP
and KCNE proteins. As each of these consists
of several isoforms and each pore forming
tetramer may interact with different combinations
of auxiliary subunits, further Kv current
heterogeneity is derived from interactions with
auxiliary subunits.1,2
Kv channels and auxiliary subunits participate
in the regulation of many cellular processes that
form the basis of normal functioning of literally
every physiological system. These range from
regulation of the heartbeat and neuronal coding
to fluid and salt secretion in epithelial tissues
such as the kidney and inner ear. Kv channels
were also implicated as key regulators of cell fate,
either as an obligatory element in the progression
of apoptosis3 or as a control element in cell cycle
progression and cellular proliferation.4,5
KCNE Subunits Structure
and Naming
KCNE are membrane-spanning proteins that
interact with ion channels and modify their
properties. Their structure consists of a single
membrane-spanning α helix, and extra- and
intracellular N and C termini, respectively (figure
1B). Five human genes were identified and
denoted KCNE1-5. The origin of this name stems
from the realization that KCNE proteins are Kv
channel auxiliary subunits and therefore these
proteins are part of the Kv channel superfamily
(all human genes of this family begin with KCN).
The first isoform to be characterized was KCNE1
(IsK), which was originally named MinK standing
for minimal K+ channel. Subsequently, the related
isoforms were named Mink Related Peptides or
MiRPs.2
Physiological Relevance of
KCNE Subunits
Several mutations in KCNE genes lead to
disease. Long QTs (LQT) are syndromes in
which cardiac arrhythmias may occur due to
prolongation of the cardiac action potential.
This cellular phenomenon is, in many cases, a
result of reduction in the magnitude of the K+
efflux, which facilitates repolarization of the cell
membrane and action potential termination. In
addition to inherited mutations in pore forming Kv
channels, mutations in KCNE1 and 2 were found
in patients with these cardiac syndromes. Such
observations emphasize the importance of KCNE
auxiliary subunits in the overall regulation of K+
homeostasis and the cellular mechanisms that
depend on it. This is further illustrated by other
mutations, causing deafness (KCNE1) or disturbed
muscle function (KCNE3).2
The KCNE auxiliary subunits regulate Kv channel
Figure 1:
A
B
Extracellular
Schematic representation of the
Lipid membrane
Intracellular
28
C
membrane topology of Kvα (A),
KCNE(B) and a channel multimer (C).
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Expression of KCNE2 in
mouse heart
Immunohistochemical staining of KCNE2 (MIRP1) in mouse
heart, using Anti-KCNE2 (MIRP1) antibody, (#APC-054).
Immunoreactivity (red) appeared in striate pattern in heart
muscle.
activities in many ways, including enhancement or
depression of permeation properties and change
in gating characteristics. However, in many cases
the modulatory action of KCNE on Kv channels is
complex, and may combine an impact on the pore
and voltage sensing properties.2
Knockout of the KCNE1 gene in mice resulted in
phenotype alterations that hint to the involvement
of these gene products in cellular volume
regulation in epithelial cells,6 a task that is
Western blotting of rat heart
membranes
Western blotting of rat heart
membranes
1. Anti-KCNE2 (MiRP1) antibody
1. Anti-KCNE1 (IsK) antibody
(#APC-054) (1:200).
(#APC-008) (1:200).
2. Anti-KCNE2 antibody, preincubated
2. Anti-KCNE1 antibody, preincubated
with the control peptide antigen.
with the control fusion protein.
common to many K+ channels and is also related
to apoptosis.3
KCNE Subunit Interactions
By using ion channel toxins and antibodies,
the stoichiometry of Kv channel interaction
with KCNE subunits was defined as 2 KCNE per
functional channel (i.e. a Kv tetramer)7 (figure 1C).
In addition, studies pointed out that the KCNE
C-terminus (intracellular) interacts with the pore
region of the Kv α subunit.8,9
Originally, MinK (KCNE1) was shown to associate
with Kv7.1 (KCNQ1 or KvLQT), and this channel’s
subunit composition was suggested as
corresponding for part of the delayed rectifier K+
current in cardiac tissue.10,11 Indeed, as mentioned
before, mutations in both genes result in very
similar pathological symptoms (i.e. LQT).1
Subsequently, other known KCNE isoforms,
MiRP1 and MiRP2 were shown to modulate other
components of the cardiac and skeletal K+ current.
MiRP1 (KCNE2) associates and suppresses the
other cardiac delayed rectifier component carried
out via Kv11.1 (HERG) channels,12 as well as the
transient (A type) component associated with
Kv4.2.13
MiRP2 (KCNE3) enhances the activity of the
skeletal muscle Kv3.4 channel. Mutations in
this gene were found in some patients with
familial periodic paralysis. It was suggested that
disrupted KCNE3 causes reduced K+ currents at
KCNE Subunits and Their Interactions with Kv and Kv-like Pore Forming Subunits
Gene
Main tissue distribution
MinK, IsK
Expressed in heart, lung, kidney, testis,
ovaries, small intestine, and peripheral
blood leukocytes.
Not detected in pancreas, spleen,
prostate and colon.
Restrictively localized in the apical
membrane portion of epithelial cells
Kv3.1: inhibitory- slower activation, stronger inactivation
Kv3.2: inhibitory- slower activation
Kv4.3: increased current
Kv7.1: slower activation, increased conductance
Kv11.1: increased current
Kv3.1: IP from co-transfected CHO cells
Kv3.2: IP from co-transfected CHO cells
Kv4.3: electrophysiology
Kv7.1: IP from co-transfected Sf9 cells
Kv11.1: IP from co-transfected CHO cells
Inherited and
drug induced LQT,
deafness
MiRP1
Highly expressed in brain, heart,
skeletal muscle, pancreas, placenta,
kidney, colon and thymus.
A small but significant expression is
found in liver, ovary, testis, prostate,
small intestine and leukocytes.
Very low expression, nearly
undetectable, in lung and spleen.
Kv3.1: inhibitory- slower activation, stronger inactivation
Kv3.2: inhibitory- slower activation
Kv4.2: inhibitory but enhances at Vrest due to slow inactivation
Kv4.3: increased current
Kv7.1: losing voltage dependence: increased current at Vrest
Kv11.1: inhibitory
HCN1: increased current.
HCN2: increased current.
HCN4: increased current.
Kv3.1: IP from co-transfected CHO cells
Kv3.2: IP from co-transfected CHO cells
Kv4.2: IP from co-transfected oocytes
Kv4.3: electrophysiology
Kv7.1: IP from co-transfected COS-7 cells
Kv11.1: electrophysiology
HCN1: IP from co-transfected oocytes
HCN2: IP from cardiomycytes
HCN4: Yeast 2 Hybrid
Inherited and
drug induced LQT
(and other cardiac
arrhythmias)
KCNE3
MiRP2
Widely expressed (skeletal muscle,
brain, heart) with highest levels in
kidney and moderate levels in small
intestine
Kv2.1: inhibitory
Kv3.1: inhibitory
Kv3.2: inhibitory
Kv3.4: enhancing current at rest.
Kv7.1: losing voltage dependence: increased current at Vrest
Kv7.4: inhibitory
Kv11.1: inhibitory
Kv2.1: IP from rat brain
Kv3.1 IP from rat brain
Kv3.2: IP from co-transfected CHO cells
Kv3.4: IP from co-transfected COS cells
Kv7.1: electrophysiology
Kv7.4: electrophysiology
Kv11.1: electrophysiology
Familial periodic
paralysis
KCNE4
MiRP3
Predominantly expressed in embryo and
adult uterus.
Low expression found in kidney, small
intestine, lung and heart.
Kv1.1: inhibitory
Kv1.3: inhibitory
Kv7.1: slower activation
KCNE5
MiRP4
Highly expressed in heart, skeletal
muscle, brain, spinal cord and placenta
Kv7.1: inhibitory
KCNE1
KCNE2
Interaction with channel and effect
Identified by
Associated
disease
Protein
Kv1.1: electrophysiology
Kv1.3: electrophysiology
Kv7.1: electrophysiology
Kv7.1: electrophysiology
AMAE
IP = immunoprecipitation.
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29
Expression of Kv4.2 in rat
hippocampus
Immunohistochemical staining of rat hippocampus with
Anti-Kv4.2 antibody (#APC-023). Note that the molecular
layers were stained while the mossy fiber terminal region was
the other KCNE isoforms had no such an effect on
HCN4 channels.20
KCNE proteins are expressed in the brain where
KCNE3 was shown to associate with and inhibit
the K+ currents carried via Kv2.1 and Kv3.1
channels.22 These results are in line with the
observation that Kv3.1 (and Kv3.2) currents are
slowed down once the pore-forming gene is coexpressed with KCNE1, KCNE2 or KCNE3.23
When tested for its modulatory activity on Kv1
channels (the Shaker subfamily), KCNE4 was
identified as an inhibitory subunit of Kv1.1 and
Kv1.3 channels. It had no apparent effect on other
Kv1 channels, suggesting that KCNE4 channels
specifically interact with these two Kv isoforms.24
Western blotting of rat heart
membranes
1. Anti-Kv7.1 antibody (#APC-022)
(1:200).
2. Anti-Kv7.1, antibody preincubated
with the control peptide antigen
not stained.
resting membrane potential, leading to membrane
depolarization. Interestingly, it was shown that
this association reduced the ability of a peptidyl
toxin BDS II to block the Kv3.4 currents, hence,
demonstrating that the pharmacology of native
channel complexes may differ from that of the
cloned pore forming α subunit.14
It is interesting to note that while KCNE1 endows
Kv7.1 with clear, slow voltage dependent
activation10,11 (similar results were obtained with
KCNE415 and KCNE516), the association of these
pore-forming channels with KCNE217 and KCNE318,
results in currents that lack voltage dependency
and may serve as “leak” K+ channels. Regarding
the specificity of the interactions it should be
noted that KCNE5 was found to modulate Kv7.1
currents with no major influence on other Kv7
combinations or on Kv11.1 channels.16
In experimental expression systems, KCNE
proteins were shown to interact with and influence
a number of Kv and related channels (See Table).
The physiological significance of these reported
interactions is not always clear and not all of these
interactions were detected in vivo.
In a number of studies, KCNE2 was implicated
as an auxiliary subunit of the Kv related HCN
channels.19-21 Hyperpolarization activated
channels (HCN) are similar in structure to the
archetypal Kv channel. However, while all other
voltage dependent channels tend to open in
response to membrane potential depolarization,
HCN channels open upon membrane potential
hyperpolarization. In turn, channel activation
results in a net cation influx, which depolarizes
the membrane potential, back to rest and beyond.
These characteristics equip the cells that express
HCN channels with the molecular machinery that
is needed for pacing the membrane potential.
Hence, the KCNE2 isoform plays a critical role in
the generation of cells with pacing capabilities,
such as the S-A node in the heart and in
pacemaker neurons. It is important to note that
30
KCNE subunits were shown to be an important
factor in determining the excitability
characteristics and capabilities of cardiac-,
skeletal-, smooth-25 muscle and neuronal cells.
The patterns of protein associations described
above, highlight the necessity of fine-tuning in K+
handling for “proper” physiological functioning.
Recently, the expression of several KCNE isoforms
was demonstrated in several uterine cancer cell
lines.26 In addition, mice carrying a truncation
mutation in Kv7.1 channel, the major known
partner of KCNE, had increased susceptibility to
gastric cancer development.27 Such observations
may stress the role played by ion channels in
general and KCNE auxiliary subunits in particular,
in the cellular mechanisms leading to cancer.
Acknowledgments
We thank Dr. B. Attalli from The Tel Aviv University
for his comments.
Inhibition of Kv3.4 channels using BDS-II-a Kv3.4 blocker
Inhibition of Kv 3.4 channels expressed in Xenopus oocytes. Left: example traces before (red) and during (black) bath perfusion
of 1 µM BDS-II (#B-450). Holding potential was –100 mV, test potential to 0 mV (100 ms) was delivered every 10 seconds.
Recordings were made while perfusing ND96 Buffer. Right: time course for the experiment shown on the left. The vertical bar
indicates the period of BDS-II perfusion.
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22. McCrossan, Z. A. et al. (2003) J. Neurosci. 23, 8077.
Anti-Kv3.2
Anti-Kv3.4
Anti-Kv4.2
Anti-Kv4.3
Anti-Kv7.1
23. Lewis, A. et al. (2004) J. Biol. Chem. 279, 7884.
1. Yu, F. H. and Catterall, W. A. (2004) Sci. STKE. 2004, re 15.
24. Grunnet, M. et al. (2003) Biophys. J. 85, 1525.
2. Abbott, G. W. and Goldstein, S. A. N. (2001) Mol. Interven. 1, 95.
25. Ohya, S. et al. (2002) Am. J. Physiol. 282, G277.
3. Meir, A. (2003) Modulator 18, 2. http://www.alomone.com.
26. Suzuki, T. and Takimoto, K. (2004) Int. J. Oncol. 25, 153.
4. Bronstein-Sitton, N. (2002) Modulator 17, 4. http://
27. Elso, C. M. et al. (2004) Hum. Mol. Genet. 13, 2813.
Toxins
www.alomone.com.
5. Bogin, O. (2003) Modulator 18, 24. http://www.alomone.com
6. Lock, H. and Valverde, M. A. (2000) J. Biol. Chem. 275, 34849.
7. Chen, H. et al. (2003) Neuron 40, 15.
8. Melman, Y. F. et al. (2004) Neuron 42, 927.
9. Romey, G. et al. (1997) J. Biol. Chem. 272, 16713.
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10. Barhanin, J. et al. (1996) Nature 384, 78.
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11. Sanguinetti, M. C. et al. (1996) Nature 384, 80.
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12. Smith, P. L. et al. (1996) Nature 379, 833.
13. Zhang, M. et al. (2001) Circ. Res. 88, 1012.
14. Abbott, G. W. et al. (2001) Cell 104, 217.
15. Teng, S. et al. (2003) Biochim. Biophys. Res. Commun. 303, 808.
16. Angelo, K. et al. (2002) Biophys. J. 83, 1997.
17. Tinel, N. et al. (2000) EMBO J. 19, 6326.
18. Schroeder, B. C. et al. (2000) Nature 403, 196.
19. Yu, H. et al. (2001) Circ. Res. 88, e84.
20. Decher, N. et al. (2003) Pflugers Arch. Eur. J. Physiol. 446, 633.
21. Qu, J. et al. (2004) J. Biol. Chem. 279, 43497.
Anti-HCN1
Anti-HCN2
Anti-HCN4
Anti-KCNE1
Anti-KNCE2
Anti-Kv1.1
Anti-Kv1.3
Anti-Kv11.1
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Anti-Kv2.1
Anti-Kv3.1b
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Anti- sloβ1 (KCNMB1)
sloβ1 is a member of a family of regulatory β subunits
tone and hypertension. Studies in sloβ1 knockout mice
We are pleased to present a new antibody directed
that control the activity of the large conductance Ca2+ -
have shown that loss of this subunit results in systemic
against the intracellular N-terminus of the rat sloβ1.
activated K+ channel KCa1.1. The family includes four
hypertension. Conversely, a recent study showed that
Anti- sloβ1 (KCNMB1) antibody (#APC-036) will also
members with a shared topology: two trans-membrane
a commonly found gain-of-function sloβ1 variant has a
recognize sloβ1 from human, mouse, dog and rabbit
domains, short intracellular N- and C-termini and a
protective effect against human diastolic hypertension.
origin.
large extracellular region. The four members of the
family have a distinct tissue distribution with sloβ1
expressed almost exclusively in smooth muscle.
Western blotting of rat
smooth muscle lysate:
1. Anti- sloβ1 antibody
(#APC-036) (1:200).
2. Anti- sloβ1 antibody,
preincubated with the
control peptide antigen.
Functionally, slo β1 increases the sensitivity of the
pore-forming KCa1.1 subunit to Ca
2+
and voltage and
it also changes its pharmacology. In the past few
years there has been much interest regarding the
role of the sloβ1 subunit in the regulation of vascular
Anti- sloβ4 (KCNMB4)
Functionally, slo β4 increases the sensitivity of the
Anti- slo β4 (KCNMB4) (#APC-061) will also recognize
pore-forming KCa1.1 subunit to Ca2+ and voltage and it
sloβ4 from human and mouse origin.
also changes its pharmacology. It has been shown that
co-expression of sloβ4 with KCa1.1 makes the latter
resistant to nM concentrations of the well-known
inhibitors Charybdotoxin (#RTC-325) and Iberiotoxin
(#RTI-400). The physiological significance of slo β4
expression in the CNS is not clear, but KCa1.1 channels
are likely involved in the regulation of neurotransmitter
release in presynaptic terminals.
We are pleased to introduce a new antibody directed
against the intracellular N-terminus of the rat sloβ4.
Modulator No.19 Spring 2005 www.alomone.com
Western blotting of rat
brain membranes:
1. Anti- sloβ4 antibody
(#APC-061) (1:200).
2. Anti- sloβ4 antibody,
preincubated with the
control peptide antigen.
31