From the Laboratory of Toxicology, University of Leuven, Van Evenstraat 4, 3000 Leuven, Belgium
Received for publication, October 18, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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An important step toward understanding the
molecular basis of the functional diversity of pacemaker currents in
spontaneously active cells has been the identification of a gene family
encoding hyperpolarization-activated cyclic nucleotide-sensitive
cation nonselective (HCN) channels. Three of the four gene
products that have been expressed so far give rise to pacemaker
channels with distinct activation kinetics and are differentially
distributed among the brain, with considerable overlap between some
isoforms. This raises the possibility that HCN channels may coassemble
to form heteromeric channels in some areas, similar to other
K+ channels. In this study, we have provided evidence
for functional heteromerization of HCN1 and HCN2 channels using a
concatenated cDNA construct encoding two connected subunits. We
have observed that heteromeric channels activate several-fold faster
than HCN2 and only a little slower than HCN1. Furthermore, the voltage
dependence of activation is more similar to HCN2, whereas the cAMP
sensitivity is intermediate between HCN1 and HCN2. This phenotype shows
marked similarity to the current arising from coexpressed HCN1 and HCN2 subunits in oocytes and the native pacemaker current in CA1
pyramidal neurons. We suggest that heteromerization may increase
the functional diversity beyond the levels expected from the number of
HCN channel genes and their differential distribution.
The newly cloned hyperpolarization-activated cyclic
nucleotide-sensitive cation nonselective
(HCN)1 ion channels give rise
to currents that are almost indistinguishable or very similar to native
pacemaker currents termed If in heart (1) and
Ih or Iq in brain tissue
(2). To date, three of the four HCN genes have been
functionally expressed, giving rise to pacemaker currents with distinct
gating properties (for review see Refs. 3-5). HCN1 channels display
fast gating properties and are almost unaffected by cAMP (6), similar
to Ih recorded from some hippocampal pyramidal
cells. HCN2 channels activate slowly and are strongly modulated by cAMP
(7, 8), similar to native pacemaker currents in some cardiac cells.
HCN4 channels activate very slowly and also respond strongly to cAMP
(9, 10), similar to Ih in thalamic relay neurons.
Expression patterns have confirmed that HCN1 is expressed restrictively
in neurons of the neocortex, hippocampus, cerebellar cortex, and
brainstem nuclei (6, 11, 12). In contrast, HCN2 is widely expressed
throughout the brain with prominent labeling of thalamic and brainstem
nuclei as well as in heart tissue (6, 7). Finally, HCN4 is
predominantly expressed in thalamus, heart, and testis (9). This
distinct distribution pattern of HCN isoforms across the brain has been
suggested to attribute to functional heterogeneity of native neuronal
pacemaker currents (12). Nevertheless, considerable overlap was
observed between HCN1 and HCN2 using in situ hybridization,
namely in hippocampal pyramidal cells and some brainstem nuclei (12).
Moreover, the coexistence of HCN1 and HCN2 mRNA has been
demonstrated at the single-cell level for hippocampal CA1 neurons (13),
raising the possibility that heteromeric channel complexes could form
in some areas.
The functional heteromerization of K+ channels with a
tetrameric subunit stoichiometry has previously been demonstrated for voltage-gated K+ channels (14-19) as well as inwardly
rectifying K+ channels (20, 21) but is currently unknown
for HCN channels. HCN channel subunits have an overall structure
similar to voltage-gated K+ channel subunits, including 6 transmembrane domains (S1-S6), a selectivity filter containing the GYG
motif, and a putative voltage sensor (S4) containing a positively
charged residue at every third position. Based on these structural
similarities it has been suggested that HCN channels will most likely
adopt the general tetrameric subunit architecture of both 6- and
2-transmembrane segment K+ channels (4). To investigate the
possibility that functional heteromeric HCN channels may form by
coassembly of different HCN isoforms, we expressed a concatenated
cDNA construct encoding a covalently linked HCN1 and HCN2 subunit
into Xenopus oocytes. Currents were recorded using the
2-microelectrode voltage clamp technique and were compared with the
biophysical properties of channels formed upon coexpression of HCN1 and
HCN2 subunits and channels formed upon expression of HCN1 and HCN2
alone. The cAMP sensitivity of the homomeric and heteromeric HCN
channels was assessed by coexpression of a G-protein-coupled receptor.
cDNA Constructs and in Vitro Transcription--
The entire
coding sequence for the mouse HCN1 and HCN2 channels (originally termed
mBCNG-1 and mBCNG-2, respectively) were subcloned into the vector
pSD64TF and pGEMHE, respectively. Plasmids were first linearized either
with BamHI (for HCN1) or with SphI (for HCN2).
Next, the cRNAs were synthesized from the linearized plasmids using the
large scale SP6 (for HCN1) or T7 (for HCN2) mMessage mMachine
transcription kit (Ambion). A concatenated construct encoding a
covalently linked HCN1 and HCN2 subunit was engineered by digesting
HCN1 with PshAI (blunt) and SpeI. HCN2 was
digested with SmaI (blunt) and SpeI and ligated
into the corresponding sites of the previous construct. The ligation
product, HCN1·HCN2, was linearized with XbaI for
size verification on an agarose gel and in vitro
transcription using the large scale SP6 mMessage mMachine transcription
kit. The encoded dimer lacks the last 45 residues of the HCN1 C
terminus and contains a 9-amino acid linker (GDPNSPFLA) that connects
HCN1-Gln865stop to HCN2-M1 (see Fig.
1A).
The human µ-opioid receptor (MOR) cDNA was subcloned into our
custom high expression vector, pGEMHE (17) and linearized for
transcription as previously described (22). The rat
G Expression and Electrophysiological Recordings--
The
isolation of Xenopus laevis oocytes was as previously
described (17). Oocytes were injected with 10 ng/50 nl HCN1, HCN2, or
HCN1·HCN2 cRNA. For determination of cAMP sensitivity, oocytes were
coinjected with 0.05 ng of MOR; 0.05 ng of G Determination of cAMP Sensitivity--
In this study, we
coexpressed either µ-opioid or 5-HT4(a) receptors with
HCN channels. Both receptors are representative members of the
G-protein-coupled receptor superfamily that display an overlapping
distribution pattern with HCN1 and HCN2 isoforms in the brain (24, 26)
and activate adenylyl cyclase (23, 24). The pathway through which the
effect occurs involves G Data Analysis--
Current-voltage relationships were obtained
by measuring the current amplitude after application of 4 s
hyperpolarizing test pulses and normalization to the fully activated
current at In this study, we investigated the possible heteromerization of
HCN channels by expressing a concatenated cDNA construct encoding a
covalently linked HCN1 and HCN2 subunit (Fig.
1A) into Xenopus oocytes. Both HCN1 and HCN2 are members of the HCN gene family (3, 5,
6), encoding pacemaker channels with easily distinguishable kinetic
properties. For comparison, all records shown in Fig. 1B
were evoked using the same voltage step protocol. Homomeric HCN1
channels activate relatively fast, whereas homomeric HCN2 channels
activate more slowly (Fig. 1B, Table
I). Expression of cRNA encoding the
dimeric construct gives rise to currents that activate somewhat slower
than HCN1, but still several-fold faster than HCN2 (Fig. 1B,
Table I). This finding provides important evidence that the
concatenated construct encodes heteromeric HCN1·HCN2 channels,
because the unexpectedly fast kinetics cannot be accounted for by the
mathematical average expected from homomeric HCN1 and HCN2 channels.
Furthermore, the heteromeric phenotype shows marked resemblance to
currents that were evoked from oocytes coinjected with equal amounts of
HCN1 and HCN2 cRNA (Fig. 1B). This result suggests that HCN1
and HCN2 subunits could spontaneously coassemble to form a population
of channels with a subunit arrangement similar to the heteromeric
channels formed by HCN1·HCN2 dimers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
sQ227L cDNA clone (23) was subcloned in
pcDNA1.1 and linearized with XbaI for in
vitro transcription. The mouse 5-HT4(a)R cDNA
clone (24) in its original vector (pRK5) was first subcloned into the
XbaI and HindIII sites of pSGEM, a modified
version of pGEMHE (25). For in vitro transcription,
5-HT4(a)R/pSGEM, was linearized with NheI. The
capped cRNAs were synthesized from the linearized plasmids using the
large-scale T7 mMessage mMachine transcription kit.
sQ227L or
0.01 ng of 5-HT4(a)R; and 10 ng of HCN1, HCN2, or
HCN1·HCN2 (final injection volume, 50 nl). Injected oocytes were
maintained in ND-96 solution (KCl 2 mM, NaCl 96 mM, MgCl2 1 mM, CaCl2
1.8 mM, HEPES 5 mM, pH 7.5) supplemented with
50 µg/ml gentamicin sulfate. Whole-cell currents from oocytes were
recorded as previously described (22). Experiments were carried out
using a high potassium (HK) external solution (KCl 96 mM,
NaCl 2 mM, MgCl2 1 mM,
CaCl2 1.8 mM, HEPES 5 mM, pH 7.5).
Reversal potentials were determined in both HK and low potassium
(ND-96) external solution (NaCl 96 mM, KCl 2 mM, MgCl2 1 mM, CaCl2
1.8 mM, HEPES 5 mM, pH 7.5). Cesium sensitivity
of all the channel constructs was assessed by application of a
hyperpolarizing pulse to
100 mV, long enough to achieve a fully
activated state (4 s for HCN1 channels, 15 s for HCN2 channels,
and 4 s for tandem HCN1·HCN2 channels).
activation of adenylyl cyclase (27, 28)
and requires coexpression of G
sQ227L (a constitutively
active mutant) in the case of the µ-opioid receptor (23). The effect
of the 5-HT4(a) receptor, in part caused by its
constitutive activity (24), occurs directly through G
s
activation. HCN currents were recorded before, during, and after
application of an agonist (1 µM DAMGO for µ-opioid
receptors and 1 µM 5-HT for 5-HT4(a)
receptors) to activate the receptor.
110 mV (HCN1),
115 mV (HCN1·HCN2 tandem), and
125 mV
(HCN2). Boltzmann activation curves were constructed using peak tail
current amplitudes observed upon application of subsequent
hyperpolarizing test pulses. Current values were plotted as a function
of the applied voltage step and fitted with a Boltzmann equation,
where I represents the current,
Ioff the offset, Imax the
maximal current, Vtest the applied test voltage,
V1/2 the midpoint potential, and S the
slope of the Boltzmann curve. Curves of n experiments were
normalized to the maximal tail current amplitude and corrected for the
offset. Time constants of activation were determined by fitting the
late rising phase of the current with a double exponential function.
The initial lag of the rising phase was excluded from the fitting
procedure to obtain an appropriate fit. The lag phase maximally
comprised the first 12 ms, 32 ms, and 280 ms of the rising current for
HCN1, HCN1·HCN2 dimers, and HCN2, respectively. Averaged data are
indicated as mean ± S.E. Statistical analysis of differences
between groups was carried out with Student's t test, and a
probability of 0.05 was taken as the level of statistical significance.
(Eq. 1)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Current traces recorded from oocytes
expressing homomeric and heteromeric HCN channels. A,
schematic illustration of the HCN1·HCN2 tandem construct.
B, comparison of pacemaker currents evoked from oocytes
expressing homomeric HCN1, homomeric HCN2 channels, and heteromeric
channels formed by tandem-linked HCN1·HCN2 subunits or by coinjection
of HCN1 and HCN2 cRNA (4-s test potentials range from 40 to
110 mV
in 10 mV steps; holding potential and tail potential were
40 mV).
Scale bars indicate 1 µA and 1 s in all panels.
C, current-voltage relationships constructed from
experiments in A for homomeric HCN1 (
), homomeric HCN2
(
) and tandem HCN1·HCN2 channels (
). D,
determination of the steady-state midpoint potential of activation
(V1/2) by application of hyperpolarizing test pulses with
increasing duration. The same symbols were used as in C. Dashed line indicates the average between
triangles and squares. Unfilled
triangles are data from Ref. 12.
Comparison between HCN clones and Ih in CA1 hippocampal neurons
Current-voltage relationships were constructed from the records in Fig.
1B and are compared in Fig. 1C. It should be
noted that the 4-s test pulses applied to record the traces shown in Fig. 1B do not allow HCN2 channels to obtain a steady-state
level of activation because of their relatively slow time course of activation (12). To make a valid comparison of the voltage dependence of activation between homomeric and heteromeric HCN1 and HCN2 channels,
we calculated half-maximal values of activation
(V1/2) from experiments with voltage steps of
increasing duration (Fig. 1D). These results show that the
use of relatively short hyperpolarizing pulses shifts the estimates of
the V1/2-values to more negative potentials.
Comparison of calculated V1/2-values shows that heteromeric
HCN1·HCN2 channels activate at membrane potentials intermediate to
homomeric HCN1 and HCN2 channels for pulses 5 s. However, heteromeric
HCN1·HCN2 channels activate at membrane potentials more negative than
the average of steady-state V1/2-values for
homomeric HCN1 and HCN2 channels (average indicated with a dashed
line), for pulses long enough to achieve steady-state levels of
activation (>10 s). This result would suggest that a
voltage-dependent transition in the opening of the
heteromeric HCN1·HCN2 channel complex is limited by the isoform that
activates at the most negative membrane voltages, as in the case
of HCN2.
As could be observed from the current traces in Fig. 1B,
heteromeric HCN1·HCN2 channels activate with a faster time course than the average of homomeric HCN1 and HCN2 channels predicts. For
clarification, we have superimposed traces from homomeric and
heteromeric channels recorded during a hyperpolarizing step to 90 mV
(Fig. 2A). The current trace
recorded from oocytes coexpressing HCN1 and HCN2 channels perfectly
matches with the trace obtained from the tandem HCN1·HCN2 channels,
suggesting that channels are formed with a subunit stoichiometry
similar to the defined subunit arrangement of the tandem-linked
channels. Next, we quantified the kinetics of activation by fitting
current traces during hyperpolarizing voltage steps (as shown in Fig.
1B) with two exponential functions (12). The calculated fast
and slow time constants of activation were plotted as a function of the
applied test potential and are shown in Fig. 2, B and
C, respectively. Values calculated for the traces in Fig.
2A were
fast = 214 ± 20 ms
(n = 6) and
slow = 909 ± 54 ms
(n = 6) for tandem HCN1·HCN2 channels and
fast = 168 ± 8 ms (n = 6) and
slow = 944 ± 49 ms (n = 6) for
coexpressed HCN1 and HCN2 channels. Homomeric HCN1 and HCN2 channels as
well as heteromeric HCN1·HCN2 channels have slow time constants of activation that are 5- to 10-fold higher than fast time constants. Compared with the respective time constants for homomeric HCN1 channels, homomeric HCN2 channels have fast and slow time constants that are ~10-fold higher. These results are in accordance with recently reported data on pacemaker channels in the mouse CNS (12).
Heteromeric HCN1·HCN2 channels have time constants of activation that
are significantly faster than the average of homomeric HCN1 and HCN2
channels predicts (indicated with a dashed line). Heteromeric HCN1·HCN2 channels activate similarly to homomeric HCN1 channels, with fast and slow time constants of activation that are
only about 3-fold higher. This result suggests that HCN1 subunits
dominate the activation rate of the heteromeric channel complex.
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Another striking feature that discriminates between homomeric HCN1 and
HCN2 channels is their differential sensitivity to cAMP (6, 7).
Hormones and neurotransmitters acting through the second messenger cAMP
finely tune the activation of the pacemaker current by shifting the
voltage dependence of activation along the voltage axis (1, 2). The
underlying mechanism involves direct binding of cAMP to the channel
cyclic nucleotide binding domain (29). Similar to studies using excised
inside-out patches (6, 7), we found that activation of a coexpressed
G-protein-coupled receptor, either the 5-HT4(a) or
µ-opioid receptor, shifts the V1/2-value by 14.9 mV toward more positive potentials for homomeric HCN2 channels and no
shift for homomeric HCN1 channels (Fig.
3A). Intriguingly, heteromeric
HCN1·HCN2 channels display a cAMP sensitivity that is intermediate to
homomeric HCN1 and HCN2 channels, with a 7.2 mV shift toward more
positive potentials in response to activation of coexpressed
G-protein-coupled receptor. Representative current traces from
experiments using homomeric and heteromeric channels coexpressed with
µ-opioid or 5-HT4(a) receptors are shown in Fig. 3,
B and C, respectively. Based on the structural
similarities of the 6 transmembrane domains, the pore-forming region
and cytoplasmic N and C termini of HCN channels with other
K+ channels, it has previously been suggested that HCN
channels will most likely conform to the tetrameric subunit arrangement of both the 6- and 2-transmembrane domain K+ channels (4).
With respect to our results with HCN1·HCN2 tandems, this idea would
suggest that functional channels are likely to be formed by coassembly
of two HCN1·HCN2 tandems, adopting a HCN1/HCN2/HCN1/HCN2 or
HCN1/HCN2/HCN2/HCN1 subunit stoichiometry. Our experimental results on
the cAMP sensitivity of heteromeric HCN1·HCN2 channels support the
idea of a tetrameric subunit arrangement, because the shift of the
V1/2-value for heteromeric HCN1·HCN2 channels (7.2 mV) is exactly half of the shifts determined for homomeric HCN1 (0.2 mV) and HCN2 (14.9 mV) channels.
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Finally, HCN channels are characterized by weak selectivity for
K+ over Na+ and the inward current is sensitive
to block by application of external Cs+. Therefore, we
compared the reversal potentials and the cesium sensitivity for
homomeric HCN1 and HCN2 channels with heteromeric HCN1·HCN2 channels.
Reversal potentials determined in ND-96 solution (2 mM
K+) and HK solution (96 mM K+) were
30 ± 1 mV (n = 5) and
2 ± 2 mV
(n = 6) for HCN1 channels,
29 ± 2 mV
(n = 5) and 0 ± 1 mV (n = 6) for
HCN2 channels, and
30 ± 2 mV (n = 6) and
1 ± 2 mV (n = 6) for tandem HCN1·HCN2 channels. Application of external Cs+ caused a complete
inhibition of the inward current through HCN channels, characterized by
IC50 values of 124 ± 28 µM
(n = 6) for HCN1 channels, 63 ± 12 (n = 6) for HCN2 channels and 79 ± 11 (n = 6) for tandem HCN1·HCN2 channels. These results
indicate that heteromeric HCN1·HCN2 channels share identical ion
selectivity and cesium sensitivity with homomeric HCN1 and HCN2 channels.
Taken together, our data raise the question about the relevance to native pacemaker currents. To address this issue, it is important to note that HCN isoforms not only differ in their biophysical properties and cAMP sensitivity, but also in their distinct distribution pattern across the brain (12, 13, 30). Nevertheless, considerable overlap was shown between HCN1 and HCN2 using in situ hybridization, namely in hippocampal pyramidal cells and some brainstem nuclei (12). Moreover, the coexistence of HCN1 and HCN2 mRNA has been demonstrated at the single cell level for hippocampal CA1 neurons (13), raising the possibility that heteromeric channel complexes could form in some areas. In Table I, we have compared our data to the results obtained by Franz et al. (13) based on the similar recording conditions (room temperature, whole cell configuration) and data analysis (double exponential fits to the time course of activation). The cAMP-induced shift of the V1/2-value for Ih in hippocampal CA1 neurons correlates well with the shift that we observed for heteromeric HCN1·HCN2 channels. This result can be expected from a population of channels that either consists of equal amounts of homomeric HCN1 and HCN2 channels or a population of channels with a heteromeric stoichiometry, similar to the tandem-linked channels. Comparison of the kinetic properties and the voltage dependence of activation for Ih in hippocampal CA1 neurons with our results however favors the formation of heteromeric channels, because equally distributed homomeric channels cannot account for the fast gating properties of the native Ih.
In conclusion, the cAMP dependence and gating properties of
tandem-linked HCN1 and HCN2 subunits reveal that HCN channels can
function as heteromers, most likely tetramers. Based on the resemblance
of the tandem it's distinct current phenotype with currents obtained
from coexpressed HCN1 and HCN2 subunits and native pacemaker current in
neurons in which different isoforms coexist, our results suggest that
heteromerization of different HCN channels may further contribute to
the functional heterogeneity of native pacemaker currents. Finally, the
dimeric construct may also serve as a model to elucidate the
physiological role of native pacemaker currents and as a relevant
pharmacological tool to develop new compounds for these channels.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. Steven A. Siegelbaum for his generous gift of mBCNG-1 and mBCNG-2 cDNAs and
for sharing his comments on our work. The µ-opioid receptor clone was
kindly provided by Dr. Lei Yu. The GsQ227L clone was a
gift from Dr. Yung H. Wong. The 5-HT4(a) receptor cDNA
was kindly donated by Dr. S. Claeysen and Dr. A. Dumuis.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Toxicology, University of Leuven, Van Evenstraat 4, 3000 Leuven, Belgium. Tel.: 32-16-323403; Fax: 32-16-323405; E-mail:
Jan.Tytgat@farm. kuleuven.ac.be.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.C000738200
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ABBREVIATIONS |
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The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective; cRNA, copy RNA; MOR, µ-opioid receptor; 5-HT4(a)R, 5-HT4(a) receptor.
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