Correspondence to: Gary Yellen, Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. Fax. (617) 432-0121; E-mail.gary_yellen@hms.harvard.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hyperpolarization-activated cation currents (Ih) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned Ih channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of 4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of Ih channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of Ih channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.
Key Words: mHCN1, SPIH, ZD7288, pore
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hyperpolarization-activated nonselective cation current (Ih)1 was first described in sinoatrial node cells of the heart, and is thought to play an important role in producing the pacemaker potential that controls the beating rate of the heart (
Many years of effort to identify the molecular nature of Ih currents have revealed a gene family that encodes Ih channels. They turned out to be related both to voltage-gated K+ channels and to cyclic nucleotidegated channels. The first member of the gene family encoding the Ih channel was mBCNG1 (now termed mHCN1;
Because Ih current contributes to the pacemaker potential in the heart sinoatrial node cells, drugs that inhibit Ih channels may be therapeutically useful in the treatment of certain cardiac arrhythmias and ischemic heart disease. The drug ZD7288 is a bradycardic agent that selectively blocks the cardiac pacemaker current, If () of
4, which was higher than expected for a direct effect of voltage on the blocker. We suspect that this voltage dependence arises from preferential binding to certain conformations visited during voltage-dependent gating. We also tested the effect of ZD7288 on SPIH, a channel cloned from sea urchin testis with characteristics similar to mammalian Ih channels (
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of Recombinant Ih Channels
For channel expression, we used the mHCN1 channel (10 mV without changing any other gating properties. The shift probably reduces the number of open Ih channels in the transfected cells in culture: open Ih channels appear to kill the cultured cells. Therefore, we used this mutant as a wild-type SPIH channel throughout the experiments. The channel cDNA was subcloned into the GW1-CMV expression vector (British Biotechnology). Human embryonic kidney 293 cells (HEK 293; American Type Culture Collection) were transiently transfected with expression plasmid containing mHCN1 or SPIH cDNA (40 µg in 200-µl cell suspension) using electroporation. The channel expression plasmid was cotransfected with the
H3-CD8 plasmid (
subunit of the human CD8 lymphocyte antigen. Cells expressing the CD8 antigen were identified visually by decoration with antibody-coated beads (
Construction of Chimeras and Site-directed Mutagenesis
Several chimeras were constructed between mHCN1 and SPIH channels using native or introduced enzyme sites. The nucleotide sequences of the chimeras were verified by sequencing. The structures of the chimeras are shown in Table 1. Point mutations were introduced by PCR (
|
Solutions and Electrophysiological Recordings
All experiments were done with excised inside-out patches (
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Block of mHCN1 by ZD7288
mHCN1 channels were expressed in HEK293 cells at levels sufficient to permit recording of currents from excised inside-out patches (Fig 1 A). The channels showed slow activation (hundreds of milliseconds) upon hyperpolarization. The G-V relationship was determined by measuring the initial tail currents at +30 mV after steps to various voltages. Fitting a Boltzmann function (Fig 1 B) gave an approximate midpoint voltage (V1/2) of -101 mV and slope of e-fold/7.1 mV, corresponding to an effective gating valence (z) of
3.6. These properties are similar to those originally reported for this clone expressed in Xenopus oocytes (V1/2 = -99.9 mV, slope = 6.0 mV;
|
We tested the effect of various concentrations of ZD7288 on mHCN1 channels at a single voltage (Fig 2 A). The blocker was applied to the exposed intracellular face of inside-out patches for 4 s, after the channels were fully opened at -110 mV (Fig 2 A). The current was reversibly blocked in a dose-dependent manner. Most of the current recovered within 1 s. Although there was a also slow component of recovery (>5 s), complete recovery was achieved between trials using a series of four 800-ms step pulses to -140 mV. The fraction of blocked current measured at the end of each application is plotted in Fig 2 B. The data were fitted with a Kd of 41 µM.2 When the blocker was applied instead to the extracellular side of the channels, it took a long time (in minutes) to get the blocking effect (data not shown), indicating that the block occurs from the intracellular side of the channels, as previously suspected from whole-cell studies (
|
Block of mHCN1 Is Voltage-dependent and Requires Channel Opening
The blocking effect of ZD7288 on mHCN1 had a surprisingly strong voltage dependence (Fig 3A and Fig B). The channels could be blocked effectively at voltages producing low levels of activation (-90 and -100 mV), but the blockade was reduced at more negative voltages, at which the channels were fully opened. To quantify this voltage dependence, the fraction blocked was fitted by a Boltzmann equation. (In this case, failure to achieve complete equilibration may lead us to overestimate the limiting degree of relief of blockade at negative voltages, or to overestimate the steepness of the voltage effect, or both.) The effective valence value (z) derived from the steepness was 4.2, which was a value comparable to that for gating of mHCN1 (3.6) obtained from the G-V. This very high z
value cannot be explained by the intrinsic voltage dependence of ZD7288, whose charge is +1. Instead, the steep voltage dependence might be related to the gating of the channels. In particular, it seemed plausible that the steep voltage dependence was due to preferential closed state blockade.
|
Earlier experiments on Ih in cardiomyocytes and neurons showed some relief of block on hyperpolarization (
ZD7288 Blockade of SPIH
The sea urchin Ih channel (SPIH) was also expressed by transfection in HEK293 cells (Fig 4 A). SPIH showed rapid inactivation at hyperpolarized voltages. Intracellular application of cAMP (100 µM) removed this inactivation and increased the peak current level by 10-fold. Fig 4 B shows the voltage dependence of the relative open probability of SPIH channels in the presence of cAMP. The characteristics are quite comparable to the properties originally reported for this clone with excised inside-out patches (
|
ZD7288 readily blocked the SPIH channels (Fig 5 A). (Unless otherwise indicated, further experiments with SPIH were done in the presence of 100 µM cAMP to produce maximal activation of the channels.) Some fraction of the current recovered rapidly from the blockade, but a portion was irreversibly blocked and did not recover, even with prolonged hyperpolarization to -160 mV (data not shown).
|
We determined the rate of onset of irreversible blockade at different voltages, by tracking the current during a series of brief applications (Fig 5 B). The dots indicate the current during monitoring pulses applied every 4 s; at each arrow, ZD7288 was applied for 1 s during an activating pulse (inset), in this case to -110 mV. After each application, there was a step reduction in the size of the current, and the current remaining after each application was plotted to determine the rate constant of irreversible blockade (Fig 5 C). Rates of irreversible blockade determined at different voltages varied in parallel with the G-V of SPIH (Fig 5 D), implying that this process requires channel opening. In the absence of cAMP, the rate was >20 times slower than that with cAMP (Fig 5 D, triangle). This too supports the idea that ZD7288 can reach its binding site only in the open state of the channels. As seen for the reversible blockade of mHCN1, there was also some reduction of blockade at the most negative voltages.
The S6 Region Is Responsible for Irreversible ZD7288 Block
mHCN1 and SPIH showed different reversibility of ZD7288 blockade, as shown by the simple comparison at the top of Fig 6. Whereas the blockade of mHCN1 recovered fully during the series of 800-ms voltage steps to -140 mV, SPIH exhibited irreversible blocker binding. After each application to SPIH, there was a step reduction in current level, and the reduction was not recovered with the series of steady pulses to 140 mV. To explore the molecular basis for this difference in the reversibility of ZD7288 blockade, we constructed chimeric channels from mHCN1 and SPIH. If a transplanted region conferred the blockade properties of the donor, then this region might be important for the binding of ZD7288. Because ZD7288 appears to act on open channels, we examined the role of the S5, P, and S6 regions, which form the pore domain. As shown in Fig 6, transplanting a relatively small portion of SPIH into the mHCN1 background was sufficient to produce irreversible binding of ZD7288 (H-S/P and H-S/S5). Because both chimeras shared the S6 region of SPIH, we focused our attention on this region.
|
Three Residues in the S6 Region Are Critical for Irreversible ZD7288 Blockade
To pinpoint the residues responsible for the reversibility, we compared the amino acid sequences of SPIH and mHCN1 (Fig 7). Overall, they showed high homology. In the lower part of S6, which is expected to contribute to the cytoplasmic entrance to the pore, three amino acids are different: residues Y355, M357, and V359 in mHCN1 correspond to F456, L458, and I460 in SPIH. Therefore, we focused on these residues as candidates for determining the reversibility of the blocker effect. Our study of the mutants at these residues was limited by the fact that not all combinations yielded functional expression. In fact, only triple mutants gave good measurable currents.
|
We examined the effect of ZD7288 on mutant mHCN1-3, which had the three SPIH residues substituted in the mHCN1 background (F, L, and I replacing Y, M, and V). Remarkably, this triple mutant of mHCN1 showed irreversible blockade comparable to that for SPIH itself (Fig 6, bottom left). Therefore, we next studied the inverse mutant, with the three mHCN1 residues substituted into the SPIH background (F456Y, L458M, and I460V; named SPIH-
3). Surprisingly, the triple mutations in SPIH resulted in a completely reversible blockade (Fig 6, bottom right). These results imply that the three residues in SPIH are critical for the irreversible blocker binding. These mutations did not produce drastic changes in the gating parameters of the channels (Table 2), arguing against an indirect effect on blockade through altered gating.
|
Trapping of ZD7288 in the SPIH-3 Mutant
Our earlier results suggested that blocker might be bound preferentially to closed channels, but that blocker could not enter closed channels. The clear prediction is that blocker should also be unable to exit a closed channel; i.e., blocker should become trapped. This idea is difficult to test in wild-type channels. For wild-type mHCN1, the kinetics of blocker dissociation and channel opening are too similar to be distinguished, while for wild-type SPIH, blockade is irreversible, making it impossible to measure dissociation. Fortunately, we could test for trapping using the triple mutant, SPIH-3. This mutant acquired completely reversible blockade like mHCN1, while preserving the fast activation of the parent channel SPIH.
To determine whether ZD7288 was trapped in this channel, we examined the rates for recovery from blockade using two different pulse protocols (Fig 8). In each case, ZD7288 was applied for 1 s to achieve nearly complete blockade. In one case (Fig 8, top), we observed recovery from blockade ( = 3.1 s) after removing the blocker and keeping the voltage at -110 mV. In the other case (Fig 8, bottom), when the blocker was removed, we immediately returned the voltage to +10 mV hoping to close the channels and trap the blocker. The voltage was held for 5 s at +10 mV. If the channels closed and trapped the blocker, there should be no recovery during this 5 s; if they do not, there should be substantial recovery. The voltage was stepped back to -110 mV to test whether recovery had occurred. There was no rapidly activated current, as would have been expected for unblocked (recovered) channels. The early current was completely blocked, and all of the current recovered at the normal rate, as though all of the channels remained blocked at the start of the pulse. Thus, no recovery had occurred during the 5-s step to +10 mV, showing that the blocker cannot exit from a closed channel.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Voltage Dependence of ZD7288 Block
The most remarkable property of ZD7288 blockade of mHCN1 channels is its steep voltage dependence, corresponding to approximately four elementary charges moving through the entire transmembrane field. It has been previously demonstrated that the blocking effect of ZD7288 is relieved by hyperpolarization ( of 5.3 (similar to z
4.2 measured here for mHCN1).
This steep voltage dependence cannot be explained easily by intrinsic voltage dependence of the blocker, because the net charge of ZD7288 is only +1. In multi-ion channels, movement of the blocker in the pore can be coupled to the movement of permeant ions, leading to a higher voltage dependence ( for the relief of blockade still seems too high to be explained by this mechanism. On the other hand, the value for the voltage dependence of blockade agrees roughly with that of the gating process, with z
3.6. To account for the steep voltage dependence of ZD7288 blockade in terms of mHCN1 gating, we propose two possible models; one with preferential closed state block (but no direct binding to the closed state), and the other with two open states having different blocker affinities. The preferential closed state blockade model is suggested by the observations that the opening of the channel is required for the block and that the blocker can be trapped in closed state of the channel (Scheme 1). At the less hyperpolarized voltages, the blocker binds weakly to the open state, but the bound blocker favors closing and stable trapping of the blocker. Once the channel closes with the blocker bound inside the pore, reopening of the channel would be much more difficult than opening of the channel without blocker. However, at more negative voltages, the blocked channels are driven into the lower affinity open blocked state. Thus, the blocker effect appears to be less efficient at the more hyperpolarized voltages. Fig 9 summarizes our data for voltage-dependent block of mHCN1; the solid lines are provided by fits to this model with preferential closed state blockade. The midpoint (V1/2) of the OB
CB transition was -130 mV, which is
20 mV more negative than the V1/2 (-112 mV) of the O
C transition, i.e., binding of the blocker stabilizes the closed state.
|
Another possible model with a similar behavior has multiple open states with different affinities for the blocker (Scheme 2). We assume that there are two open states (of similar conductance) with voltage-dependent switching between them; both states are blocked by ZD7288 but with different affinities. Opening of the channels by hyperpolarization allows ZD7288 to bind with a high affinity to the O1 state. However, further hyperpolarization drives mHCN1 channels to another open state (O2), and this conformational change either prevents ZD7288 from binding or reduces its affinity substantially. Thus, blocker potency decreases at more hyperpolarized voltages. The dashed lines in Fig 9 indicate the fits according to this model with two open states, where O1 binds blocker with a Kd of 6.4 µM and O2 has much lower affinity (Kd = 1.3 mM).
Both models can mimic the experimental observations, but it is difficult to decide between them. Our observation of blocker trapping demands some form of closed state blockade; on the other hand, prominent delays in deactivation kinetics provide a clear indication of multiple open states (our unpublished observation). The correct description will probably incorporate features of both models.
Blockers that appear to bind in the pore but have a closed state preference have been described for other channels. High affinity binding of tetracaine to cyclic nucleotidegated channels occurs only with the closed conformation of the pore (
Comparison with Previous Observations
Previous work suggested that Ih blockade by ZD7288 does not require prior opening of channels. The different result seen here may be simply a difference in the variety of the h-channel studied, or it may be because of different experimental conditions. All previous experiments on ZD7288 blockade were done in a whole-cell mode with continuous application of blocker, and it took a long time (minutes to tens of minutes) to achieve blockade (
It is possible that the results showing nonuse-dependent block of ZD7288 in the previous experiments were due to slow access of the blocker to the closed channel via a hydrophobic pathway (20 times slower than with maximal activation (with 100 µM cAMP).
Previous observations on other blockers of Ih channels such as UL-FS 49 and DK-AH 268 have shown use-dependent onset of blockade (
The Site of ZD7288 Blockade
The characteristics of ZD7288 blockade appear to be compatible with binding of the compound in the pore. The blocker enters mainly or exclusively when the channel is open, and can be trapped in a closed channel. Moreover, substantial changes in blockade can be produced by mutations in S6, which is known from work on the related potassium channels to contribute to the pore (
If the three S6 residues swapped between mHCN1 and SPIH are indeed critical for direct binding, then hydrophobic interaction may dominate the binding of the blocker to the channel. The combined mutations F456Y, L458M, and I460V in SPIH make the channel less hydrophobic, and these eliminate irreversible block by ZD7288. Remarkably, the inverse mutations in mHCN1 are sufficient to confer irreversible blocker binding. The strong effect of these mutations is partly explained because each mutation is present in all four subunits of the channel (assuming that like its molecular relatives, the functional Ih channel is a tetramer). Although the individual exchange mutations were not tolerated, we have found that substitution of cysteine at one of the three positions (SPIH 460, corresponding to Shaker position 474, facing the pore) is sufficient to allow completely reversible blockade of SPIH (data not shown).
Although there are indications of ZD7288 trapping in mHCN1 and SPIH, detailed experiments were prohibited either by slow activation of mHCN1 or by irreversible blockade of SPIH. The SPIH-3 mutant acquired completely reversible blockade like mHCN1, while preserving the fast activation of its parent SPIH. The clear-cut distinction between opening and unblock made it easy to observe trapping in this mutant. Trapping of blocker in channels was first proposed for quaternary ammonium compounds in the potassium channels of squid giant axon (
If ZD7288 indeed binds in the section of the pore lined by this part of S6, the behavior of the blocker suggests that the Ih activation gate may reside in the region below these residues (i.e., more intracellular). Of course, ZD7288 is much larger than the permeant ions Na+ and K+, so it remains possible that such an intracellular gate regulates access of the blocker, while another gate (e.g., in the selectivity filter) regulates ion flow. The three S6 residues identified here are located in a homologous position to the water-filled cavity of the KcsA channel (KcsA positions 103, 105, and 107;
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: 4-AP, 4-aminopyridine; Ih, hyperpolarization-activated nonselective cation current.
2 The failure of the blockade to reach steady state, because of the slow second phase of blockade, means that the measured dissociation constant reported here is an upper limit and the true affinity for long applications may be higher.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Drs. U. Benjamin Kaupp and Gareth Tibbs for sharing their clones with us. We also thank Dr. Bruce Bean for helpful comments on the manuscript, members of our lab for discussion, and Tara Ogren and Tanya Abramson for transfected cells.
This work was supported by grants to G. Yellen from the National Institutes of Health (HL57383) and the McKnight Endowment Fund for Neuroscience.
Submitted: 2 November 2000
Revised: 5 December 2000
Accepted: 5 December 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Armstrong, C.M. 1971. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axon. J. Gen. Physiol. 58:413-437
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 1996. Current Protocols in Molecular Biology. New York, John Wiley & Sons Inc. Section 8.5.
Berger, F., Borchard, U., Gelhaar, R., Hafner, D., and Weis, T.M. 1995. Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres. Naunyn-Schmiedeberg's Arch. Pharmacol. 353:64-72[Medline].
BoSmith, R.E., Briggs, I., and Sturgess, N.C. 1993. Inhibitory action of ZENECA ZD 7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br. J. Pharmacol. 110:343-349[Abstract].
Brown, H., and DiFrancesco, D. 1980. Voltage-clamp investigations of membrane currents underlying pace-maker activity in rabbit sinoatrial 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].
DiFrancesco, D. 1981. A new interpretation of the pace-maker current in calf Purkinje fibres. J. Physiol. 413:359-376.
DiFrancesco, D. 1986. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature. 324:470-473[Medline].
DiFrancesco, D. 1993. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55:455-472[Medline].
DiFrancesco, D. 1994. Some properties of the UL-FS 49 block of the hyperpolarization-activated (if) in sino-atrial node myocyte. Pflügers Arch. 427:64-70.
DiFrancesco, D., and Tortora, P. 1991. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature. 351:145-147[Medline].
Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., and MacKinnon, R. 1998. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77
Fodor, A.A., Black, K.D., and Zagotta, W.N. 1997. Tetracaine reports a conformational change in the pore of cyclic nucleotidegated channels. J. Gen. Physiol. 110:591-600
Frace, A.M., Maruoka, F., and Noma, A. 1992. External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells. Pflügers Arch. 421:97-99.
Gasparini, S., and DiFrancesco, D. 1997. Action of the hyperpolarization-activated current (Ih) blocker ZD7288 in hippocampal CA1 neurons. Pflügers Arch. 435:99-106.
Gauss, R., Seifert, R., and Kaupp, U.B. 1998. Molecular identification of a hyperpolarization-activated channel in sea urchin sperm. Nature. 393:583-587[Medline].
Hagiwara, S., Miyazaki, S., and Rosenthal, N.P. 1976. Potassium current and effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J. Gen. Physiol. 67:621-638[Abstract].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Harris, N.C., and Constanti, A. 1995. Mechanism of block by ZD 7288 of the hyperpolarization-activated inward rectifying current in guinea pig substantia nigra neurons in vitro. J. Neurophysiol. 74:2366-2378
Hille, B. 1977. Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor interaction. J. Gen. Physiol. 69:497-515[Abstract].
Hille, B., and Schwarz, W. 1978. Potassium channels as multi-ion single-file pores. J. Gen. Physiol. 72:159-162.
Holmgren, M., Smith, P.L., and Yellen, G. 1997. Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating. J. Gen. Physiol. 109:527-535
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
Janigro, D., Martenson, M.E., and Baumann, T.K. 1997. Preferential inhibition of Ih in rat trigeminal ganglion neurons by an organic blocker. J. Membr. Biol. 160:101-109[Medline].
Jurman, M.E., Boland, L.M., Liu, Y., and Yellen, G. 1994. Visual identification of individual transfected cells for electrophysiology using antibody-coated beads. Biotechniques. 17:876-881[Medline].
Kirsch, G.E., and Drewe, J.A. 1993. Gating-dependent mechanism of 4-aminopyridine block in two related potassium channels. J. Gen. Physiol. 102:797-816[Abstract].
Liu, Y., Holmgren, M., Jurman, M.E., and Yellen, G. 1997. Gated access to the pore of a voltage-dependent K+ channel. Neuron. 19:175-184[Medline].
Ludwig, A., Zong, X., Jeglitsch, M., Hoffmann, F., and Biel, M. 1998. A family of hyperpolarization-activated mammalian cation channels. Nature. 393:587-591[Medline].
Ludwig, A., Zong, X., Stieber, M.J., Hullin, R., Hoffmann, 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., Bal, T., and McCormick, D.A. 1998. Periodicity of thalamic spindle waves is abolished by ZD7288, a blocker of Ih. J. Neurophysiol. 79:3284-3289
Maccaferri, G., and McBain, C.J. 1996. The hyperpolarization-activated current (Ih) and its contribution to pacemaker activity in rat CA1 hippocampal stratum oriens-alveus interneurones. J. Physiol. 497:119-130[Abstract].
Mayer, M.L., and Westbrook, G.L. 1983. A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones. J. Physiol. 340:19-45[Abstract].
McCormack, K., Joiner, W.J., and Heinemann, S.H. 1994. A characterization of the activating structural rearrangements in voltage-dependent Shaker K+ channels. Neuron. 12:301-315[Medline].
McCormick, D.A., and Bal, T. 1997. Sleep and arousal: thalamocortical mechanisms. Annu. Rev. Neurosci. 20:185-215[Medline].
Mitcheson, J.A., Chen, J., Lin, M., Culberson, C., and Sanguinetti, M.C. 2000. A structural basis for drug-induced long QT syndrome. Proc. Natl. Acad. Sci. USA. 97:12329-12333
Pape, H.C. 1996. Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu. Rev. Physiol. 58:299-327[Medline].
Pape, H.C., and McCormick, D.A. 1989. Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current. Nature 340:715-718[Medline].
Raes, A., van de Vijver, G., Goethals, M., and van Bogaert, P.P. 1998. Use-dependent block of Ih in mouse dorsal root ganglion neurons by sinus node inhibitors. Br. J. Pharmcol. 125:741-750[Abstract].
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., 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., Shan, 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
Satoh, T., and Yamada, M. 2000. A bradycardic agent ZD7288 blocks the hyperpolarization-activated current (Ih) in retinal rod photoreceptors. Neuropharmacol. 39:1284-1291[Medline].
Seed, B., and Aruffo, A. 1987. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA. 84:3365-3369[Abstract].
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
Spassova, M., and Lu, Z. 1998. Coupled ion movement underlies rectification in an inward-rectifier K+ channel. J. Gen. Physiol. 112:211-221
Strata, F., Atzori, M., Molnar, M., Ugolini, G., Tempia, F., and Cherubini, E. 1997. A pacemaker current in dye-coupled hilar interneurons contributes to the generation of giant GABAergic potentials in developing hippocampus. J. Neurosci. 17:1435-1446
Van Bogaert, P.P., Goethals, M., and Simoens, C. 1990. Use- and frequency-dependent blockade by UL-FS 49 of the If pacemaker current in sheep cardiac purkinje fibres. Eur. J. Pharmacol. 187:241-256[Medline].
Williams, S.R., Turner, J.P., Hughes, S.W., and Crunelli, V. 1997. On the nature of anomalous rectification in thalamocortical neurons of the cat ventrobasal thalamus in vitro. J. Physiol. 505:724-747.
Wollmuth, L.P. 1995. Multiple ion binding sites in Ih channels of rod photoreceptors from tiger salamanders. Pflügers Arch. 430:34-43.
Yanagihara, K., and Irisawa, H. 1980. Inward current activated during hyperpolarization in the rabbit sinoatrial node cell. Pflügers Arch. 385:11-19.
Yellen, G. 1998. The moving parts of voltage-gated ion channels. Q. Rev. Biophys. 31:239-295[Medline].
Yu, H., Chang, F., and Cohen, I.S. 1993. Pacemaker current exists in ventricular myocytes. Circ. Res. 72:232-263[Abstract].