doi: 10.1161/hh1201.093511
© 2001 American Heart Association, Inc.
UltraRapid Communication |
MinK-Related Peptide 1
A ß Subunit for the HCN Ion Channel Subunit Family Enhances Expression and Speeds Activation
From the Institute of Molecular Cardiology (H.Y., J.W., J.Z., Z.P., H.W., W.S., M.R.E.-M., W.B., J.D., D.M., I.S.C.), Department of Physiology & Biophysics (H.Y., J.W., I.P., J.Z., H.W., W.S., M.R.E.-L., W.B., J.D., I.S.C.), and Department of Neurobiology & Behavior (Z.P., D.M.), SUNY at Stony Brook, Stony Brook, NY; Department of Biology (R.W., R.T.W., B.H.), University of Tulsa, Tulsa, Okla; and Department of Pharmacology (R.B.R.), Columbia University, New York, NY. Current address for H.Y. is Department of Physiology, New York Institute of Technology, Old Westbury, NY.
Correspondence to Dr Ira S. Cohen, Department of Physiology & Biophysics, 8661 SUNY, Stony Brook, NY 11794-8661. E-mail icohen{at}physiology.pnb.sunysb.edu
Abstract
Abstract—The HCN family of ion channel subunits underlies the currents If in heart and Ih and Iq in the nervous system. In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a ß subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a ß subunit for the cardiac delayed rectifier IKr, these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org.
Key Words: HCN family • MiRP1 • KCNE family • ß subunit
The HCN
(hyperpolarization-activatedcyclic
nucleotide–gated) family of ion channel subunits has been identified as the molecular
correlate of the currents
If in
heart and
Ih and
Iq in
neurons.1 2 3
However, several ion channels are heteromultimers of a large
subunit (like the HCN family members) and smaller ß subunits. The
cardiac delayed rectifiers
IKr4
and
IKs5
are examples of this basic principle. Their subunits derive from
the ERG and KCNQ families, respectively, but both also contain ß
subunits from the KCNE family of single transmembrane–spanning
proteins called minK and minK-related peptides (MiRPs). In this study,
we report that MiRP1 enhances the expression and speeds the kinetics of
activation of the HCN family of channel subunits. From
immunoprecipitation experiments, we show that it most probably forms a
complex with HCN1. Using RNase protection assays (RPAs), we demonstrate
that MiRP1 mRNA is prevalent in the primary cardiac pacemaking region,
the sinoatrial (SA) node, and barely detectable in ventricle. Cardiac
pacemaker activity is generated by a narrow balance of inward
(If) and
outward
(IKr)
currents. Our results demonstrate for the first time the potential
importance of a single ß subunit in simultaneously
regulating both the expression and gating of both inward and outward
cardiac pacemaker channels.
Materials and Methods
Heterologous Expression in
Xenopus Oocytes
cRNA encoding mouse HCN1 or HCN2, rat MiRP1 with or
without an HA tag at the carboxy-terminal, and rat minK were
transcribed using the mMessage mMachine kit
(Ambion). Xenopus
laevis oocytes were isolated, injected with 2 to 5 ng (50 to
100 nL) of cRNA, and maintained in Barth medium at 18°C for 1 to 3
days. For experiments using both HCN1 or HCN2 and MiRP1 or minK, the
respective cRNAs were injected in a 1:0.04 to 1 ratio.
Electrophysiological studies on oocytes used the
2-microelectrode voltage clamp. The extracellular recording
solution (OR2) contained, in mmol/L, NaCl 80, KCl 2,
MgCl2 1, and Na-HEPES 5 (pH 7.6). Group data are
presented as mean±SEM. Tests of statistical significance for
midpoint and slope of activation curves were performed using unpaired
Student’s t tests.
P<0.05 is considered
significant.
RNase Protection Assays
The procedures for the preparation of total RNA from
rabbit heart tissues and the performance of the RNase
protection assays were essentially identical to those described
previously.6 Brain total RNA
was obtained commercially from Clontech, and
total RNA was isolated from left ventricle, right atrium, and brain
using SV Total RNA System (Promega). For each
experiment, 2 µg of total RNA was used. A commercially available rat
cyclophilin probe was used in each experiment as an internal control
over sample loss (Ambion). The undigested rabbit
MiRP1 probe is 260 nt, whereas the upper protected fragment is 210 nt
and the lower protected fragment is 
195 nt. The undigested
cyclophilin probe is 167 nt, and the protected rabbit cyclophilin band
is 80 nt. RNA expression was quantified directly from dried RNase
protection assay gels using a Storm PhosphorImager
(Molecular Dynamics) normalized to the
cyclophilin signal in each lane. The MiRP1 signal consisted of two
protected fragments in each rabbit tissue where MiRP1 was detected. The
presence of two bands is likely the result of the degenerate polymerase
chain reaction primers that were designed from mouse and human
sequences, used for the cloning of the rabbit RPA probe (GenBank No.
AF329636). The combined intensity of both bands was used in the
quantification.
Protein Chemistry
Membrane Preparation
All steps were performed in ice. Twenty-five oocytes
were washed with Ringer solution (in mmol/L, NaCl 96,
CaCl2 1.8, and HEPES 5 [pH 7.4]) and lysed by
vortexing with 1 mL lysis buffer 1 (7.5 mmol/L
Na2HPO4 [pH 7.4] and
1 mmol/L EDTA) with protease inhibitors (aprotinin,
leupeptin, and pepstatin, 5 µg/mL of each, and 1 mmol/L PMSF).
The lysate was centrifuged for 5 minutes at
150g to remove yolk proteins
and subsequently for 30 minutes at
14 000g. The membrane pellet
was washed with lysis buffer 1 and resuspended in 1 mL of lysis buffer
2 (in mmol/L, Tris-HCl 50 [pH 7.5], NaCl 150, EDTA 5, NaF 50,
sodium pyrophosphate 50,
KH2PO4 100, sodium
molybdate 10, and sodium orthovanadate 2 and 1% Triton X-100 and 0.5%
NP40) with the same set of protease inhibitors as lysis
buffer 1 followed by clarifying by centrifugation at
14 000g for 5 minutes. Protein
concentration of the membrane fractions was determined by the Lowry
method.
Immunochemistry
Samples were separated on 10%
SDS/PAGE in Tris-glycine buffer (for HCN1) or on
16.5% SDS/PAGE in Tris-tricine
buffer7 [for MIRP1(HA)] and
electroblotted to Hybond
ECL nitrocellulose membranes
(Amersham Pharmacia Biotech) or to
Immobilon-PSQ
membrane (Millipore). Western blots were
performed with rabbit anti-HCN1 antibody (Quality Controlled
Biochemicals) or rat anti-HA high affinity antibody (Roche
Molecular Biochemicals) and with a horseradish peroxidase–coupled
secondary antibody (Kirke- gaard Perry Laboratories) or
biotin-conjugated affinity-purified secondary antibody
(Chemicon International, Inc). Streptavidin-POD
conjugate was from Roche Molecular Biochemicals. The immunoreactive
bands were visualized using ECL Western blotting
detection reagents (Amersham Pharmacia
Biotech).
The immunoprecipitation reactions were performed with 750 µg of membrane protein fraction and anti-HCN1 antibody cross-linked to Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc) with dimethyl pimelimidate (Sigma).
Results
We used the
Xenopus oocyte as a
heterologous expression system and examined the expression of HCN1 and
HCN2 individually and coexpressed with either minK (the minimal
K+ channel protein, the first identified
member of the single transmembrane–spanning protein family) or MiRP1.
The results are shown in
Figure 1
. Both HCN1
(Figure 1A) and HCN2
(Figure 1D) express a small current when injected alone.
Coexpression of either HCN1
(Figure 1B) or HCN2
(Figure 1E) with minK results in similar, low levels of
current expression. However, a much larger current is observed when
either HCN1
(Figure 1C) or HCN2
(Figure 1F) is coexpressed with MiRP1. Injection of MiRP1 by
itself did not induce a current nor did injection with 100 nL of
H2O (not shown). Our complete set of results for
the expression studies of HCN1 and HCN2 with or without minK and MiRP1
is illustrated in
Figures 1G and 1H. The maximal conductance is calculated by
dividing the tail current, the result of stepping back to -10 mV from
the most negative voltage step, by the driving force (the reversal
potential was measured in each oocyte). The results demonstrate an
almost 3-fold enhancement of HCN1 conductance when HCN1 is coexpressed
with MiRP1 (P<0.01), whereas
MiRP1 enhances expression of HCN2 by more than 5-fold
(P<0.01). Coexpression of
either HCN1 or HCN2 with minK does not significantly alter HCN1 or HCN2
expression (P>0.05). Thus, the
enhancement of expression is specific for MiRP1.
|
We next examined the gating properties of MiRP1 coexpressed
with either HCN1 or HCN2. The results are presented in
Figure 2. Isochronal activation curves were constructed
from tail currents recorded at -10 mV in response to 3-second-
(for HCN1) or 8-second- (for HCN2) long hyperpolarizing test pulses.
The results demonstrate no significant difference in midpoint but
statistically indicate a shallower slope for the activation of HCN
channels coexpressed with MiRP1
(Figures 2A and 2B, see legends for details).
|
We also examined the kinetics of activation and deactivation
(see
Figures 2C
through Figure 2F). Raw data are shown for
activation of both HCN1
(Figure 2C) and HCN2
(Figure 2D). MiRP1 decreases the time constant of activation.
Statistical comparisons were made at each potential
(P<0.05). When the same
comparisons are made for deactivation (data enclosed within a box in
Figures 2E and 2F), there is no significant difference at any potential
(P>0.05) although there is a
tendency toward acceleration.
The rectification properties of HCN1 or HCN2 expressed with or without MiRP1 were also studied. Coexpression of either HCN1 or HCN2 with MiRP1 did not alter the linearity of the fully activated current-voltage relationship (not shown).
Previous studies examining the potential role of MiRP1 in
generating
IKr used
Northern blot analysis to demonstrate the presence of MiRP1
mRNA in whole rat heart.4 If
MiRP1 also regulates
If
current expression in vivo, mRNA levels for MiRP1 should be prominent
in regions where
If
currents are large. We used RNase protection assays to quantify the
distribution of MiRP1 transcripts in SA node, right atrium, and
ventricle of the rabbit heart. The results are provided in
Figure 3. MiRP1 transcript levels are highest in the SA
node, atrial levels are 40% of those in SA node, and
ventricular levels are barely detectable (<4% of SA
node).
|
To show that MiRP1 could be a ß subunit for the HCN family, we demonstrated that a complex probably exists between members of the HCN family and MiRP1. We chose to pursue this question with HCN1 because we have raised an antibody against this family member.
The HCN1 antibody reveals a single polypeptide with an
apparent molecular mass of 145 kDa (possibly
glycosylated).8 MiRP1, HA
epitope-tagged at the carboxy terminal end, was recognized by anti-HA
high-affinity antibody as a 13.5-kDa band. Both proteins were localized
in the membrane fraction, and protein expression was enhanced
(2-fold) when they were coexpressed together
(Figures 4A and 4B).
|
To test whether a complex of HCN1 and MiRP1(HA) might exist
in a heterologous expression system, we performed coimmunoprecipitation
experiments using membrane fractions of oocytes injected with HCN1
alone, MiRP1(HA) alone, or both cRNAs.
Figure 4C shows the immunoprecipitation products tested
by Western blot analysis. The presence of MiRP1(HA) in the
anti-HCN1 immunoprecipitate only for oocytes injected by both HCN1 and
MiRP1(HA) cRNAs, together with its absence in oocytes injected with one
of these cRNAs, indicates that MiRP1(HA) was pulled down by anti-HCN1
antibody most likely because it was complexed with HCN1.
Our results demonstrate that the proteins are colocalized as a complex in the membrane and enhance each other’s expression. This strongly suggests that MiRP1 is a ß subunit for the HCN family of ion channel subunits.
Discussion
MiRP1 is a member of a family of single transmembrane–spanning proteins that have been demonstrated to alter expression and serve as a ß subunit of both KCNQ (minK) and ERG (MiRP1) family members.4 5 In these previous studies, as in our study, the minK family member altered gating and was demonstrated to be a ß subunit by coimmunoprecipitation.
In this study, we have shown that minK is largely without effect on the properties of HCN1 and HCN2 channels expressed in Xenopus oocytes. MiRP1, on the other hand, dramatically enhances the current expression of both HCN subunits and hastens the kinetics of current activation. A speeding of deactivation kinetics is seen when MiRP1 associates with HERG to form IKr.4
MiRP1 is expressed in rabbit SA node at significant levels
as determined by RPA (see
Figure 3), suggesting its potential importance as a ß
subunit for both
IKr and
If in SA
node. The low level of MiRP1 expression in rabbit ventricle raises
questions about its importance in generating
IKr in
this ventricular tissue and suggests that additional
studies in the ventricle of other species would be
valuable.
It is well-known that ß subunits of Na+ and Ca2+ channels can alter both expression and gating.9 More recently, similar evidence for effects of ß subunits on K+ channel expression have also been demonstrated.10 Our results show that MiRP1 and HCN1 probably form a complex in the membrane and so add the nonspecific cyclic nucleotide–gated channels to this growing list of ion channels whose expression or gating is regulated by a ß subunit.
Pacemaker activity in the rabbit sinus node is generated by a net inward current of only a few pA.11 This net inward current is attributable to the balance of inward and outward currents more than an order of magnitude larger. Although the biophysical properties of each of the component currents is known, how this fine balance is achieved remains a mystery. Our results imply for the first time that a single ß subunit may control the expression of two important pacemaker currents, the outward IKr and the inward If. Thus, MiRP1 could serve as an important regulator of cardiac pacemaker rate.
Acknowledgments
This work was supported by grants HL28958 and HL20558 from the National Heart, Lung, and Blood Institute and Scientist Development Awards from the American Heart Association to H.Y. and R.W. We gratefully acknowledge the gift of the HCN1 and HCN2 clones from Drs B. Santoro and S. Siegelbaum.
Footnotes
Original received March 16, 2001; resubmission received May 11, 2001; revised resubmission received May 23, 2001; accepted May 25, 2001.
References
- Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR. Identification of a gene encoding a hyperpolarization activated pacemaker channel of brain. Cell. 1998;93:717–729.
- Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization activated mammalian cation channels. Nature. 1998;393:587–591.
- Santoro B, Tibbs GR. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann N Y Acad Sci. 1999;868:741–764.
- Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, Goldstein SA. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell. 1999;97:175–187.
- Sanguinetti MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, Keating MT. Coassembly of KvLQT1 and minK (ISK) proteins to form cardiac ISK potassium channel. Nature. 1996;384:80–83.
- Dixon JE, McKinnon D. Quantitative analysis of potassium channel expression in atrial and ventricular muscle of rats. Circ Res. 1994;75:252–260.
- Sclägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacriglamide gel electrophoresis for separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166:368–379.
- Hansen JE, Lund O, Engelbrecht J, Bohr H, Nielsen JO, Hansen JE. Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Biochem J. 1995;308:801–813.
- Isom LL, DeJongh KS, Catterall WA. Auxiliary subunits of voltage-gated ion channels. Neuron. 1994;12:1183–1194.
- Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS. ß Subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron. 1996;16:843–852.
- Vassalle M, Yu H, Cohen IS. Pacemaker channels and cardiac automaticity. In: Zipes D, Jalife J, eds. Cardiac Electrophysiology. From Cell to Bedside. Philadelphia, Pa: W.B. Saunders; 2000:94–103.
This article has been cited by other articles:
 |
|
 |
|
  J. Qu, Y. Kryukova, I. A. Potapova, S. V. Doronin, M. Larsen, G. Krishnamurthy, I. S. Cohen, and R. B. Robinson MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes J. Biol. Chem., October 15, 2004; 279(42): 43497 - 43502. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||