Correspondence to: Steven A. Siegelbaum, Center for Neurobiology and Behavior, Columbia University, 722 West 168 Street, New York, NY 10032. Fax:(212) 795-7997 E-mail:sas8{at}columbia.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 510-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 1020 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP doseresponse curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of Ih in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.
Key Words: potassium channel, gating, heteromultimer, Ih, cAMP
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hyperpolarization-activated cationic currents (Ih)1 were initially identified in cardiac myocytes (
The recent cloning of a family of four mammalian genes encoding hyperpolarization-activated cAMP-regulated cation (HCN) channels (
When expressed in heterologous systems, three of the four HCN genes have been shown to generate hyperpolarization-activated currents with distinct biophysical properties. HCN1 channels activate fastest and require the least amount of hyperpolarization to open (
Although the recombinant HCN channels and native Ih currents share basic properties, it has not yet been shown whether any HCN homomeric channel can fully reproduce the characteristics of any native Ih current. Since multiple HCN isoforms may be coexpressed in the same cell (
To investigate the possible formation and resultant properties of heteromeric Ih channels, we coinjected cRNAs encoding mouse isoforms of HCN1 and HCN2, which are coexpressed in neocortical and hippocampal neurons, in Xenopus oocytes. Ih generated by coexpression of HCN1 and HCN2 subunits was clearly distinct from Ih generated by homomeric HCN1 or HCN2 channels, providing strong evidence for the formation of heteromultimeric Ih channels with novel properties. In the course of these experiments, we further noticed significant differences between Ih recorded in intact oocytes versus cell-free patches. By making a point mutation in the CNBD to prevent cAMP modulation, we demonstrated that at least a part of these differences is due to the modulation of Ih in intact oocytes by basal levels of cAMP. Thus, these results suggest that properties of Ih in native neurons and cardiac cells are likely to be determined by both coassembly of distinct HCN subunits and basal modulation by resting levels of cyclic nucleotide.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Biology
Mouse HCN1 (
|
|
|
|
|
|
|
Expression in Xenopus Oocytes
RNA was transcribed from NheI-linearized DNA (HCN1) or SphI-linearized DNA (HCN2) using a T7 RNA polymerase (Message Machine; Ambion) and injected into Xenopus oocytes as described previously (
Electrophysiological Recordings
Two microelectrode voltage-clamp recordings were obtained 12 d after cRNA injection using an oocyte clamp amplifier (model OC-725B; Warner Instruments). Data were filtered at 250 Hz and sampled at 500 Hz using an ITC-18 interface and Pulse software (HEKA). The recordings were obtained with the oocytes bathed in a high KCl extracellular solution containing (in mM): 96 KCl, 2 NaCl, 10 HEPES, and 2 MgCl2, pH 7.5. Microelectrodes were filled with 3 M KCl and had resistances of 0.52 M. Holding potential was -30 mV. Analysis was done using Pulsefit (HEKA) and IgorPro (WaveMetrics).
Cell-free inside-out patches were obtained 36 d after cRNA injection, and data were acquired using a patch-clamp amplifier (model Axopatch 200A; Axon Instruments). A symmetrical solution was used containing (in mM): 107 KCl, 5 NaCl, 10 Hepes, 1 MgCl2, and 1 EGTA, pH 7.3. Patch pipets were 13 M, and were coated with Sylgard to minimize capacitance. The holding potential for these inside-out patches was -40 mV. A Ag-AgCl ground wire was connected to the bath solution by a 3-M KCl agar bridge electrode, and junction potential was compensated before the formation of each patch. Linear leak was not subtracted. Acquired data were filtered at 1 kHz with the Axopatch 200A built-in 4-pole low pass Bessel filter and sampled at 2 kHz with an ITC-18 interface. Analysis was done using PulseFit, IgorPro, and Sigma Plot.
Hyperpolarizing voltages in 10- or 5-mV step increments were applied to either inside-out patches or intact oocytes from the holding potential. All recordings were obtained at room temperature (2225°C).
Data Analysis
Steady-state activation curves were determined from the amplitude of tail currents after hyperpolarizing steps on return to -40 mV. Tail current amplitudes were measured after the decay of the capacitive transient by averaging the current during the plateau of the tail. Current values were plotted as a function of the step voltages and fit with the Boltzmann equation: I(V) = A1 + A2/{1+exp[(V - V1/2)/s]}, where A1 is an offset caused by a nonzero holding current, A2 is the maximal tail current amplitude, V is voltage during the hyperpolarizing test pulse in mV, V1/2 is the midpoint activation voltage, and s is the slope of fitting. To average the data from different experiments, the tail current amplitudes for each individual experiment were normalized by first subtracting the fitted value of A1, and then dividing by the fitted value of A2. These normalized data were averaged among the different experiments and the averaged, normalized data were then fitted by the Boltzmann equation with A1 set to 0 and A2 set to 1. These normalized curves were plotted in the indicated figures.
Activation time constants were determined by fitting the current evoked during hyperpolarizing voltage steps to single or double exponential functions using Pulsefit. Simultaneous fitting with two exponential components yielded fits that were significantly better than single exponential terms for all currents activated in response to voltages that were negative to V1/2; for all the currents including the coexpression of two HCN channels, the fit was not improved after addition of a third component. The uncompensated capacitive transients and activation delays occurring in the initial phase of the Ih currents (initial 50100 ms) were excluded from the fitting windows. Data are presented as mean ± SEM.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The properties of hyperpolarization-activated currents upon coexpression of HCN1 and HCN2 subunits were compared with the Ih currents generated upon expression of HCN1 or HCN2 alone. If the two subunits did indeed coassemble to form a heteromultimer with novel properties, we expected that the heteromultimeric Ih would not be adequately described by the algebraic sum of two independent populations of HCN1 and HCN2 channels, at any proportional ratio.
In Intact Oocytes, Coexpression of HCN1 and HCN2 Generates an Ih with Novel Properties
We first characterized properties of Ih generated by expression of HCN1 alone, HCN2 alone, or coexpression of the two isoforms in intact oocytes using two microelectrode voltage-clamp. As reported previously (
The time course of Ih upon coexpression of HCN1 and HCN2 subunits could not be reproduced by the algebraic sum of independent populations of homomeric HCN1 and HCN2 channel currents, suggesting the formation of heteromultimeric Ih currents with distinct properties (Fig 1 B). To characterize the properties of the coexpressed channels, we fit the time course of Ih activation with two exponential components (Fig 2 A), which were necessary and sufficient to describe adequately the activation kinetics of the coexpressed currents as well as the kinetics of HCN1 or HCN2 homomultimers (
For all recombinant Ih studied, the fast and slow exponential components (f and
s) were voltage-dependent, speeding up at more hyperpolarized voltages. Over the entire voltage range of activation, the fast and slow time constants of activation for HCN1 were
10-fold more rapid than the respective time constants for HCN2 (
The voltage dependence of the relative amplitudes of the fast and slow exponential components differs significantly between HCN1 and HCN2 homomeric channels (80%) of the current amplitude, and this proportion did not depend on the voltage during the hyperpolarization. In contrast, for HCN2 channels, the slow component was predominant for relatively small hyperpolarizations, where less than half the channels open. At more hyperpolarized voltages, the contribution of the fast component for HCN2 became progressively greater. The relative amplitude of the fast and slow exponential components of Ih generated by coexpression of HCN1 and HCN2 showed a marked dependence on voltage that was similar to, but slightly less steep than, the behavior of HCN2 channels (Fig 2 B).
Examination of the steady-state voltage dependence of channel activation further supported the view that HCN1 and HCN2 subunits formed heteromultimeric channels. Tail current activation curves were measured for Ih generated by expression of HCN1 alone, HCN2 alone, and coexpression of HCN1 and HCN2 (Fig 3). As shown previously (
Coexpression of HCN1 and HCN2 Studied in Inside-out Patches: Novel Gating and Modulation by cAMP
The effects of cAMP on HCN channel function were measured using cell-free inside-out patches, which permitted the rapid application of solutions to the internal face of the membrane. As previously reported for Ih in cardiac myocytes (3s.
The effects of application of a saturating concentration of cAMP were studied for Ih generated by HCN1 alone, HCN2 alone, and by coexpression of HCN1 and HCN2 (Fig 4B and Fig C). Similar to previous findings, cAMP caused only a small increase in the rate of activation of HCN1 channels (
The effects of cAMP on the voltage dependence of gating were examined next using tail current activation curves (Fig 5 and Table 1). In the absence of cAMP, HCN1 activated at voltages that were 20 mV more positive than those required to activate HCN2 channels. For HCN1, the V1/2 of activation was -115.8 ± 1.3 mV with a slope of 6.3 ± 0.7 mV. For HCN2, the V1/2 was -135.7 ± 1.7 mV with a slope of 4.3 ± 0.3 mV. Thus, although V1/2 values were shifted by 50 mV relative to their values in intact oocytes, the qualitative difference in voltage dependence between HCN1 and HCN2 was maintained in the inside-out patches. In fact, the 20-mV difference in V1/2 between HCN1 and HCN2 in cell-free patches was larger than the 10-mV difference observed in intact oocytes.
|
Channels generated by the coinjection of HCN1 and HCN2 showed an intermediate voltage dependence of activation, with a V1/2 of -129.7 ± 1.1 mV and a slope of 4.4 ± 0.5 mV (Fig 5 A and Table 1). This result is somewhat surprising given the results presented above that, in intact oocytes, the V1/2 of the coexpressed channels was similar to that of HCN1, not intermediate between HCN1 and HCN2. The explanation for this discrepancy, as well as the greater difference in V1/2 between HCN1 and HCN2 homomeric channels in inside-out patches versus intact oocytes, is explored below.
Further evidence that Ih generated by coexpression of HCN1 and HCN2 subunits reflected the novel properties of heteromeric channels was provided by comparison of experimental and simulated tail current activation curves (Fig 5). The activation data for Ih measured in patches from oocytes in which HCN1 and HCN2 were coexpressed (either for Ih from a single, representative patch [ Fig 5 B], or averaged from seven separate patches [ Fig 5 C]) could not be accounted for by the sum of activation curves for independent populations of HCN1 and HCN2 channels at varying proportions.
Because of the quantitative difference in the response of HCN1 versus HCN2 homomeric channels to cAMP, we next examined the effect of this nucleotide on the gating of the coexpressed HCN channels (Fig 5 and Table 1). As previously shown, a saturating concentration of cAMP (10 µM) shifted the V1/2 of HCN1 channels by only 4 mV (similar to the findings of
17 mV (similar to the findings of
Doseresponse relations for the shift in V1/2 as a function of [cAMP] were compared for HCN1, HCN2, and coexpressed channels (Fig 6). The doseresponse curves were fitted by the Hill equation to obtain the maximal shift at saturating [cAMP], the cAMP concentration at which half of the maximal shift was produced (K1/2), and the Hill coefficient (h). For HCN2 channels, the maximal shift with cAMP was 17.4 mV with a K1/2 of 0.10 µM (h = 1.1). For HCN1 channels, the shift was only 4.1 mV with a K1/2 of 0.06 µM (h = 1.0). Thus, the small effect of cAMP on HCN1 gating does not reflect a low sensitivity to the ligand. Finally, for channels formed by coexpression, the maximal shift was 14.0 mV with a K1/2 of 0.19 µM (h = 1.3). Surprisingly, the K1/2 for channels generated by coexpression was greater than the K1/2 for either of the homomeric channels. Furthermore, the doseresponse curve for the coexpressed channels could not be reproduced by the sum of independent populations of homomeric HCN1 and HCN2 channels (Fig 6).
Modulation of HCN Channels by Basal cAMP in Intact Oocytes Studied through an Inactivating Point Mutation in the Cyclic Nucleotide Binding Domain
Although the above results in intact oocytes and cell-free patches supported the view that HCN1 and HCN2 subunits coassemble to form heteromultimeric channels with novel properties, there were certain puzzling differences in the behavior of the various channels in the two recording configurations. First, we found a large, 50-mV hyperpolarizing shift in V1/2 values measured for Ih in cell-free patches relative to values in intact oocytes. Moreover, we found a larger difference in V1/2 values between HCN1 channels and HCN2 channels in cell-free patches (20 mV) than in intact oocytes (9 mV). Finally, in cell-free patches, the V1/2 of coexpressed channels was intermediate between the V1/2 values for channels formed by HCN1 or HCN2 alone. In contrast, in intact oocytes, the V1/2 for coexpressed channels was similar to that of HCN1 channels. What might account for such differences?
Given the high sensitivity of HCN channels to cAMP, we investigated whether basal levels of cAMP in the intact oocytes might have been sufficient to modulate the gating of HCN channels. To investigate this possibility, we mutated a single arginine residue (R538 in HCN1 and R591 in HCN2) that is conserved in nearly all CNBDs (
In the background of both HCN1 and HCN2 channels, the arginine (R) to glutamate (E) mutation had a very similar effect as in CNG channels. Thus, the gating of mutant HCN1/R538E and HCN2/R591E homomeric channels in inside-out patches was completely unaffected by 10 µM cAMP (Fig 7B and Fig C, and Table 1), which is a concentration that is 50100-fold higher than the K1/2 for modulation of wild-type HCN channels. However, the mutation had no effect on the intrinsic gating properties of the channels, as shown by the nearly identical activation curves of wild-type and mutant channels in the absence of cAMP (Table 1 and Fig 8B and Fig C).
|
In contrast to the lack of effect of these mutations on HCN gating in cell-free patches, in intact oocytes studied by two microelectrode voltage-clamp, we observed a pronounced negative shift in the gating of HCN1/R538E channels, HCN2/R591E channels, and coexpressed mutant channels relative to the gating of the respective wild-type channels. Thus, the V1/2 of HCN1/R538E was -75.9 ± 1.0 mV with a slope of 6.1 ± 0.3 mV (n = 10 cells), representing a shift of about -7 mV compared with wild-type HCN1 channels (Fig 8 B). The V1/2 of HCN2/R591E was -97.1 ± 0.9 mV with a slope of 5.0 ± 0.3 (n = 9), representing a shift of about -19 mV compared with wild-type HCN2 (Fig 8 C). Finally, the V1/2 of the currents from oocytes coinjected with HCN1/R538E and HCN2/R591E was -81.8 ± 1.6 mV with a slope of 6.7 ±0.3 (from 10 cells), representing a shift of -14 mV (comparing Fig 3 with Fig 8 A). These shifts in V1/2 values for HCN1/R538E (7 mV), HCN2/R591E (19 mV), and coexpressed mutant subunits (14 mV) in intact oocytes are compatible with the maximal shifts of 4, 17, and 14 mV seen in response to cAMP in inside-out patches for channels formed by the corresponding wild-type HCN channels. Thus, our results are consistent with the view that basal levels of cAMP were sufficient to cause a maximal positive voltage shift in the gating of HCN channels in intact oocytes. Differences in efficacy of cAMP in modulating HCN1, HCN2, and HCN1/HCN2 heteromultimers could explain some of the discrepancies between Ih properties recorded from inside-out patches versus intact oocytes (Fig 8B and Fig C).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our study in both intact Xenopus oocytes and inside-out patches demonstrated that HCN1 and HCN2 subunits, when coexpressed, form functional heteromultimeric channels that generate hyperpolarization-activated currents with novel properties. Furthermore, we showed that the basal level of cAMP may play an important role in the modulation of HCN channel function in intact cells. Both coassembly and basal cAMP modulation significantly increase the potential for functional diversity of Ih in the nervous and cardiovascular systems.
Formation of Heteromultimers between Two Different Isoforms of HCN Channels
In situ hybridization studies of mouse brain have revealed distinct but overlapping patterns of expression of HCN1 and HCN2 (
By coexpressing HCN1 and HCN2 in Xenopus oocytes, we have provided several lines of functional evidence that the two isoforms can indeed coassemble to form functional heteromultimers with novel properties. In intact oocytes, coexpression of HCN1 and HCN2 gave rise to Ih with a voltage dependence similar to that of HCN1 channels, but with kinetics that were twice as slow. In inside-out patches, the coexpressed channels displayed a voltage dependence and an efficacy of cAMP modulation that were intermediate between those of HCN1 and HCN2 channels. Simulation of hyperpolarization-activated currents generated by the summed contributions of independent populations of HCN1 and HCN2 channels in various ratios could not reproduce the currents we observed from coinjected oocytes. Finally, the coexpressed channels displayed a decreased sensitivity to cAMP (increased K1/2) compared with either HCN1 or HCN2 channels.
Although the hypothesis that HCN1 and HCN2 subunits coassemble to form functional heteromultimeric channels with novel properties provides the simplest explanation for our findings, a number of more complicated scenarios might be envisioned. For example, HCN1 and HCN2 could compete for some limiting cofactor in the oocytes (e.g., a ß subunit or modulatory enzyme), so that coexpression of the two subunits leads to a change in the functional properties of homomeric HCN1 and HCN2 channels, relative to their properties when expressed alone. However, the fact that the steady-state activation curves we observe upon coexpression of HCN1 and HCN2 (in both cell-free patches and intact oocytes) is as steep as that observed upon expression of either HCN1 or HCN2 alone argues strongly against the presence of two distinct channel populations (which would inevitably lead to a shallower activation curve, unless the V1/2 values just happened to coincide). Furthermore, such a competition for a limiting cofactor is inconsistent with our finding that V1/2 values are independent of level of Ih current expression (Fig 3 B). This latter finding also argues against a change in homomeric channel properties due to some direct interaction between homomeric HCN1 and HCN2 channels.
Further evidence that the properties of the channels observed upon coexpression of HCN1 and HCN2 subunits do indeed reflect the properties of heteromeric channels comes from a recent study of
The only significant quantitative discrepancy between our results and those of
The novel biophysical characteristics of the heteromultimeric channels endow them with unique potential physiological functions. Their relatively positive threshold of activation would allow them to control resting membrane properties and to help generate pacemaker potentials after repolarization of the action potential. Their pronounced modulation by cAMP would contribute to alterations in cellular excitability by hormones and transmitters. These properties of the heteromultimeric channels correspond well with the properties of certain native Ih as discussed two sections below.
Modulation by Basal Level of Cyclic Nucleotides in Cells
Based on differences in channel properties in cell-free patches versus intact cells, together with the high sensitivity of HCN channels to cAMP, we investigated the possible modulation of HCN channels by basal levels of cyclic nucleotide in the intact oocytes. Mutation of a conserved arginine in the ß roll of the cyclic nucleotide binding domain to a glutamate completely prevented the modulatory action of cAMP, without altering normal gating properties of either HCN1 or HCN2 channels in cell-free patches. At the structural level, this result is in good agreement with previous results in CNG channels, where a similar mutation blocked activation by cyclic nucleotide without altering the free energy difference between open and closed states in the absence of the ligand (
Although the point mutations had no effect on the voltage gating of HCN channels in cell-free patches, we did observe significant differences between the mutant channels and wild-type channels in intact oocytes. The voltage dependence of HCN1 and HCN2 homomeric channels was shifted in the hyperpolarized direction by 7 and 19 mV, respectively. Such shifts are nearly identical to the maximal shifts seen with the binding of saturating concentrations of cAMP to HCN1 and HCN2 wild-type channels in cell-free patches (4 and 17 mV, respectively; Fig 6). This suggests that the basal level of cAMP in oocytes is sufficient to produce near maximal shifts in gating of wild-type HCN channels. This view is compatible with the observation that the K1/2 values of these channels range from 50 to 200 nM (Fig 6), and that resting cAMP levels in oocytes can be in the micromolar range (
The modulation by basal levels of cAMP, however, accounts for only part of the difference in the V1/2 values between wild-type Ih in intact oocytes and inside-out patches. This difference is quite large, amounting to a -47-mV shift for HCN1 and a -57 mV shift for HCN2 (V1/2 in inside-out patches minus the V1/2 in intact oocytes). Similar shifts are also observed for native Ih currents in cardiac myocytes (
Coassembly Is Compatible with the Ih in Native Tissues That Express Both HCN1 and HCN2
Although our results show that HCN1 and HCN2 can efficiently coassemble to form heteromultimers in heterologous expression systems, our experiments do not prove that heteromultimer formation necessarily does occur in native tissues in which the subunits are coexpressed. Unfortunately, a lack of suitable antibodies specific for HCN1 and HCN2 isoforms precludes coimmunoprecipitation experiments. However, a careful comparison of the properties of Ih in native tissues that coexpress HCN1 and HCN2 with the properties of Ih generated by the recombinant HCN gene products reported here does indicate that coassembly in vivo is likely. In Fig 9, we show data from
|
![]() |
Footnotes |
---|
S. Chan and J. Wang contributed equally to this work.
1 Abbreviations used in this paper: CNBD, cyclic nucleotide binding domain; HCN, hyperpolarization-activated cAMP-regulated cation; Ih, hyperpolarization-activated cationic currents.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Bina Santoro, Gareth Tibbs, and Brian Wainger for their helpful discussions and assistance, Eric Odell for help in preparing the manuscript, and Huan Yao and John Riley for their technical assistance.
This work was partially supported by grant RO1 NS-36658 (to S.A. Siegelbaum) from the National Institutes of Health. In addition, J. Wang was supported by the Medical Scientist Training Program.
Submitted: 16 February 2001
Revised: 30 March 2001
Accepted: 2 April 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beaumont, V., and Zucker, R.S. 2000. Enhancement of synaptic transmission by cyclic AMP modulation of presynaptic Ih channels. Nat. Neurosci. 3:133-141[Medline].
Brown, H., and DiFrancesco, D. 1980. Voltage-clamp investigations of membrane currents underlying pace-maker activity in rabbit sino-atrial node. J. Physiol. 308:331-351[Abstract].
Brown, H.F., DiFrancesco, D., and Noble, S.J. 1979. How does adrenaline accelerate the heart? Nature. 280:235-236[Medline].
Demontis, G.C., Longoni, B., Barcaro, U., and Cervetto, L. 1999. Properties and functional roles of hyperpolarization-gated currents in guinea-pig retinal rods. J. Physiol. 515:813-828
DiFrancesco, D. 1993. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55:455-472[Medline].
DiFrancesco, D., and Tortora, P. 1991. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature. 351:145-147[Medline].
DiFrancesco, D., and Mangoni, M. 1994. Modulation of single hyperpolarization-activated channels (i(f)) by cAMP in the rabbit sino-atrial node. J. Physiol. 474:473-482[Abstract].
DiFrancesco, D., Ducouret, P., and Robinson, R.B. 1989. Muscarinic modulation of cardiac rate at low acetylcholine concentrations. Science. 243:669-671[Medline].
Franz, O., Liss, B., Neu, A., and Roeper, J. 2000. Single-cell mRNA expression of HCN1 correlates with a fast gating phenotype of hyperpolarization-activated cyclic nucleotide-gated ion channels (Ih) in central neurons. Eur. J. Neurosci. 12:2685-2693[Medline].
Goulding, E.H., Ngai, J., Kramer, R.H., Colicos, S., Axel, R., Siegelbaum, S.A., and Chess, A. 1992. Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons. Neuron. 8:45-58[Medline].
Ishii, T.M., Takano, M., Xie, L.H., Noma, A., and Ohmori, H. 1999. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J. Biol. Chem. 274:12835-12839
Jan, L.Y., and Jan, Y.N. 1997. Cloned potassium channels from eukaryotes and prokaryotes. Annu. Rev. Neurosci. 20:91-123[Medline].
Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F., and Biel, M. 1998. A family of hyperpolarization-activated mammalian cation channels. Nature. 393:587-591[Medline].
Ludwig, A., Zong, X., Stieber, J., Hullin, R., Hofmann, F., and Biel, M. 1999. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO (Eur. Mol. Biol. Organ.) J. 18:2323-2329
Luthi, A., and McCormick, D.A. 1998. H-current: properties of a neuronal and network pacemaker. Neuron. 21:9-12[Medline].
Magee, J.C. 1999. Dendritic Ih normalizes temporal summation in hippocampal CA1 neurons. Nat. Neurosci. 2:848[Medline].
Maller, J.L., Butcher, F.R., and Krebs, E.G. 1979. Early effect of progesterone on levels of cyclic adenosine 3':5'- monophosphate in Xenopus oocytes. J. Biol. Chem. 254:579-582[Abstract].
McCormick, D.A., and Pape, H.C. 1990. Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones. J. Physiol. 431:291-318[Abstract].
Monteggia, L.M., Eisch, A.J., Tang, M.D., Kaczmarek, L.K., and Nestler, E.J. 2000. Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Brain Res. Mol. Brain Res. 81:129-139[Medline].
Moosmang, S., Biel, M., Hofmann, F., and Ludwig, A. 1999. Differential distribution of four hyperpolarization-activated cation channels in mouse brain. Biol. Chem. 380:975-980.
Morozov, A., E. Gibbs, M.F. Nolan, C. Kentros, G. Malleret, B. Santoro, and E.R. Kandel. 2000. Generation and characterization of mice harboring a knockout of the hyperpolarization-activated channel HCN1. Soc. Neurosci. 803.1 (Abstr.).
Nolan, M.F., A. Morozov, E. Gibbs, S.A. Siegelbaum, and E.R. Kandel. 2000. Contribution of HCN1 channels to H-current and membrane properties of CA1 pyramidal neurons. Soc. Neurosci. 803.2 (Abstr.)
Pape, H.C. 1996. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu. Rev. Physiol. 58:299-327[Medline].
Santoro, B., and Tibbs, G.R. 1999. The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels. Ann. NY Acad. Sci. 868:741-764
Santoro, B., Grant, S.G., Bartsch, D., and Kandel, E.R. 1997. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels. Proc. Natl. Acad. Sci. USA. 94:14815-14820
Santoro, B., Liu, D.T., Yao, H., Bartsch, D., Kandel, E.R., Siegelbaum, S.A., and Tibbs, G.R. 1998. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell. 93:717-729[Medline].
Santoro, B., Chen, S., Luthi, A., Pavlidis, P., Shumyatsky, G.P., Tibbs, G.R., and Siegelbaum, S.A. 2000. Molecular and functional heterogeneity of hyperpolarization activated pacemaker channels in the mouse CNS. J. Neurosci. 20:5264-5275
Seifert, R., Scholten, A., Gauss, R., Mincheva, A., Lichter, P., and Kaupp, U.B. 1999. Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proc. Natl. Acad. Sci. USA. 96:9391-9396
Shabb, J.B., and Corbin, J.D. 1992. Cyclic nucleotide-binding domains in proteins having diverse functions. J. Biol. Chem. 267:5723-5726
Shi, W., Wymore, R., Yu, H., Wu, J., Wymore, R.T., Pan, Z., Robinson, R.B., Dixon, J.E., McKinnon, D., and Cohen, I.S. 1999. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ. Res. 85:1-6
Southan, A.P., Morris, N.P., Stephens, G.J., and Robertson, B. 2000. Hyperpolarization activated currents in presynaptic terminals of mouse cerebellar basket cells. J. Physiol. 526:91-97
Su, Y., Dostmann, W.R., Herberg, F.W., Durick, K., Xuong, N.H., Ten Eyck, L., Taylor, S.S., and Varughese, K.I. 1995. Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science. 269:807-813[Medline].
Tibbs, G.R., Liu, D.T., Leypold, B.G., and Siegelbaum, S.A. 1998. A state-independent interaction between ligand and a conserved arginine residue in cyclic nucleotide-gated channels reveals a functional polarity of the cyclic nucleotide binding site. J. Biol. Chem. 273:4497-4505
Ulens, C., and Tytgat, J. 2001. Functional heteromerization of HCN1 and HCN2 pacemaker channels. J. Biol. Chem. 276:6069-6072
Weber, I.T., and Steitz, T.A. 1987. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J. Mol. Biol. 198:311-326[Medline].
Zagotta, W.N., and Siegelbaum, S.A. 1996. Structure and function of cyclic nucleotide-gated channels. Annu. Rev. Neurosci. 19:235-263[Medline].