| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Journal of Physiology (2001), 537.1, pp. 93-100
© Copyright 2001 The Physiological Society
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were first described in the rabbit heart sinoatrial node. These channels are characterized by slow activation induced by hyperpolarization to generate inward currents, termed If in cardiac cells and Ih in neurones (DiFrancesco, 1993; Pape 1996).
Recently, four kinds of cDNAs for HCN channels were cloned in mammals (HCN1-4) (Santoro et al. 1998; Ludwig et al. 1998; Ishii et al. 1999; Ludwig et al. 1999; Seifert et al. 1999; Vaccari et al. 1999; Moroni et al. 2000; Monteggia et al. 2000). Structurally, HCN channels consist of six transmembrane domains with a pore region between S5 and S6 and a cyclic nucleotide-binding domain (CNBD) in the cytoplasmic C-terminal region, which is similar to the structure of cyclic nucleotide-gated channels (Santoro et al. 1998; Ludwig et al. 1998).
HCN1 channels are activated most rapidly by hyperpolarization (in tens of milliseconds) (Santoro et al. 1998; Santoro et al. 2000), whereas HCN4 channels are the slowest to be activated (in seconds) (Ishii et al. 1999; Ludwig et al. 1999; Seifert et al. 1999). The different activation kinetics have been implicated in different physiological roles of these HCN subtypes. In the central nervous system, HCN2 and HCN4 mRNAs were found in neurones that display prominent oscillations. HCN1 is richly expressed in the dendrites of layer V pyramidal neurones in the cerebral cortex, and is proposed to play a role in regulating signal integration by virtue of its rapid kinetics (Santoro et al. 2000).
Since the region responsible for the activation kinetics remains elusive, we decided to determine the structural basis for the differences in activation kinetics. We constructed chimeras between HCN1 and HCN4, and introduced site-directed mutations into HCN1, and the effects of these mutations were examined.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Molecular biology
HCN1 and HCN4 were cloned from mouse and rabbit, respectively. Animals were kept and killed according to the guiding principles and regulations of Kyoto University.
Mouse HCN1 cDNA was cloned from mouse brain RNA with the RT-PCR method and LA Taq (TaKaRa, Kyoto, Japan). The amino acid sequence was identical to the published sequence (Ludwig et al. 1998), except for the deletion of 15 glutamine residues out of a series of 37 glutamine residues in the C-terminal region. Mouse brain RNA was the gift of Dr M. Hazama (Kyoto University).
Rabbit HCN4 cDNA contains an N-terminal region that is longer by 25 amino acid residues than that cloned previously (Ishii et al. 1999). The 25-amino-acid sequence at the N-terminus was identical to that of human HCN4 (Ludwig et al. 1999; Seifert et al. 1999). We isolated the 5' end of rabbit HCN4 cDNA from the rabbit heart sinoatrial node RNA by using 5' RACE (SMART RACE cDNA Amplification Kit, Clontech, Palo Alto, CA, USA) and LA Taq (TaKaRa). The rabbits were deeply anaesthetized with an intra-venous injection of an overdose of pentobarbital sodium (sodium 5-ethyl-(1-methylbutyl) barbiturate; 100 mg (kg body weight)-1), and the sinoatrial node of the heart was dissected out. RNA was extracted by Trizol (Life Technologies, Inc., NY, USA).
Deletion mutants were constructed using PCR and restriction sites. We refer to the C-terminus-deleted HCN1 as 1
C and to the N- and C-termini-deleted HCN4 as 4NC (Fig. 1A). 1C lacks 289 amino acid residues at the C-terminus in mouse HCN1. 4NC lacks 214 amino acid residues at the N-terminus and 422 amino acid residues at the C-terminus in rabbit HCN4. Chimeras and site-directed mutations were created by overlap PCR using PfuTurbo (Stratagene, La Jolla, CA, USA). All PCR products were verified by sequencing (BigDye Terminator Cycle Sequencing, Applied Biosystems, Inc., Foster City, CA, USA). Oligonucleotides were from Life Technologies, Inc. The chimeric junctions were generated by overlap extension of PCR primers that encoded the desired sequence:
4-1A: 215-259 of HCN4 and 128-621 of HCN1;
4-1B: 215-283 of HCN4 and 152-621 of HCN1;
4-1C: 215-295 of HCN4 and 164-621 of HCN1;
4-1D: 215-320 of HCN4 and 189-621 of HCN1;
4-1E: 215-405 of HCN4 and 274-621 of HCN1;
4-1F: 215-555 of HCN4 and 424-621 of HCN1;
4-1G: 215-602 of HCN4 and 471-621 of HCN1;
4-1-4A: 215-259 and 296-753 of HCN4, and 128-163 of HCN1;
4-1-4-1A: 215-259 and 296-555 of HCN4,
and 128-163 and 424-621 of HCN1;
1-4A: 1-188 of HCN1 and 321-753 of HCN4;
1-4B: 1-423 of HCN1 and 556-753 of HCN4;
1-4C: 1-470 of HCN1 and 603-753 of HCN4;
1-4D: 1-503 of HCN1 and 636-753 of HCN4;
1-4E: 1-529 of HCN1 and 662-753 of HCN4;
1-4-1A: 1-127 and 164-621of HCN1, and 260-295 of HCN4;
1-4-1B: 1-273 and 371-621of HCN1, and 406-502 of HCN4;
1-4-1C: 1-370 and 424-621 of HCN1, and 503-555 of HCN4.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 1. Activation kinetics and voltage-activation curves of HCN1, HCN4 and their deletion mutants A, schematic representation of mouse HCN1, rabbit HCN4 and their deletion mutants. Black regions represent HCN1 and white regions HCN4 amino acid sequences in this and subsequent figures. B, representative current recordings of HCN1, HCN4 and their deletion mutants (1 | ||
Functional expression and electrophysiological measurements
All channel subunits and green fluorescent protein (GFP) S65A cDNA were subcloned into independent PCI vectors (Promega, Madison, WI, USA) and the mixture of vectors were transfected into COS-7 cells (RIKEN, Wako, Japan) using LipofectAMINE (Life Technologies, Inc.) as described before (Ishii et al. 1999). Currents were recorded from transfected COS-7 cells with the whole-cell patch recording technique using an EPC-7 amplifier (List, Darmstadt, Germany). Patch pipettes were made of borosilicate glass (Harvard Apparatus Ltd, Edenbridge, Kent, UK) and the pipette resistances were 4-8 M
when measured in experimental solutions. The series resistance was 5-20 M and was compensated by 60-90 %. All current records were made within 3 min of attaining whole-cell access. Data were low-pass filtered at 2 kHz, analog-to-digital (A/D) sampled at 0.1-20 ms intervals with 12 bit resolution, and stored in a personal computer (PC-9821 Ap; NEC, Tokyo, Japan). The whole-cell currents were fitted to single exponential functions except for the initial lag (see Fig. 1C). The limitations of single exponential fitting are detailed in Discussion. The membrane potential was held at -20 mV and step pulses were applied from -20 to -120 mV. Tail currents were measured at 0 mV as described before (Ishii et al. 1999). Normalized tail current amplitude was plotted versus test potential to obtain the voltage-dependent activation curve and was fitted with the Boltzmann function: Itail/Itail,max = 1/(1 + exp((Vm - V0.5)/S)), where Vm is the test potential, V0.5 is the membrane potential for the half-maximal activation, and S is the slope factor. Most experiments were performed at 25.0 ± 0.5 °C, and some experiments were performed at 35.0 ± 0.5 °C particularly when HCN4 channels and its chimeras were tested (Fig. 1 and Fig. 4). The raised temperature accelerated activation kinetics and facilitated the evaluation of voltage dependence of steady-state activation of the channel. The intracellular (pipette) solution contained (mM): 135 KCl, 5 EGTA, 5 NaCl, 10 Hepes and 5 KOH (pH 7.4). The extracellular (bath) solution contained (mM): 155 NaCl, 2.5 CaCl2, 1 MgCl2, 17 glucose, 10 Hepes and 5 KOH (pH 7.4). Data are given as means ± S.E.M. (number of experiments). Statistical differences were determined using Student's unpaired t test; P values < 0.05 were considered significant.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
HCN1 is activated about 40-fold faster than HCN4
Mouse HCN1 and rabbit HCN4 were transiently expressed in COS-7 cells and were examined by using the whole-cell patch clamp technique. Both HCN1 and HCN4 showed robust inward currents by hyperpolarizing voltage steps (Fig. 1B). Currents were usually fitted well with single exponential curves except for the initial lag (Fig. 1C). HCN1 was activated rapidly during the -100 mV hyperpolarizing voltage step (Fig. 1Ca), and the activation time constant (
) was 13.4 ± 2.0 ms (n = 6) at 35 °C (Fig. 1D). HCN4 was activated quite slowly at -100 mV (Fig. 1Cb) with a of 497 ± 31 ms (n = 10) at 35 °C (Fig. 1D). HCN1 was activated 37-fold faster than HCN4 at -100 mV (Fig. 1D).
We deleted the C-terminus of HCN1 and the N- and C-termini of HCN4 to make subcloning of chimeras and mutants easy because HCN channels contain long N- and C-termini that contain GC-rich regions. In HCN1, the deletion of the C-terminus (1C) changed neither the activation kinetics nor the voltage-activation curve significantly (Fig. 1D and Ea). However, the deletion of the N- and C-termini in HCN4 (4NC) shifted the voltage- activation curve by 10.1 mV in the hyperpolarizing direction and slowed the activation at -100 mV 1.2-fold (Fig. 1D and Eb).
HCN1-HCN4 chimeras showed various activation kinetics
The regions responsible for the differences of activation kinetics between HCN1 and HCN4 were investigated by constructing chimeras between 1C and 4NC (Fig. 2A). values during the -100 mV hyperpolarizing voltage step were determined at room temperature (25 °C) (Fig. 2B). In these measurements, no chimeras were steady-state activated at the holding potential level (-20 mV) and no tail currents were observed at 0 mV. In Fig. 2, the chimeras whose N-termini are derived from 4NC are shown (from 4-1A to 4-1G). As the region derived from HCN4 was longer, became longer. Especially, the introduction of the S1 region from HCN4 markedly prolonged (between 4-1A and 4-1B: 6.5-fold; P < 0.01). Besides, values between 4-1B and 4-1C were significantly different (1.7-fold; P < 0.01). values between 4-1F and 4-1G were also statistically different (1.6-fold; P < 0.01). These results demonstrate that the introduction of the S1-S2 linker and the cytoplasmic loop region between S6 and the cyclic nucleotide-binding domain (CNBD) affected the activation kinetics.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 2. Comparison of activation time constants among HCN1-HCN4 chimeras during -100 mV hyperpolarizing voltage steps A, schematic representation of HCN1, HCN4, their deletion mutants and HCN1-HCN4 chimeras. The exact points of the crossovers are shown in parentheses for HCN1 top and HCN4 bottom. B, activation time constants during -100 mV hyperpolarizing voltage steps for HCN channels shown in A at 25 °C. The number of experiments in each group is given in parenthesis. The V0.5 and slope factors, respectively, were -68.5 ± 2.7 mV and 8.4 ± 1.4 mV for 4-1A (n = 4), -79.5 ± 2.3 mV and 8.9 ± 0.6 mV for 4-1B (n = 6), -81.8 ± 1.2 mV and 6.2 ± 0.4 mV for 4-1C (n = 7), -69.0 ± 1.3 mV and 5.6 ± 0.5 mV for 4-1D (n = 6), -72.0 ± 1.4 mV and 5.8 ± 0.6 mV for 4-1E (n = 7), -75.3 ± 2.3 mV and 6.8 ± 0.8 mV for 4-1F (n = 7), -73.7 ± 2.5 mV and 6.7 ± 0.4 mV for 4-1G (n = 6), -68.6 ± 3.0 mV and 6.6 ± 0.5 mV for 1-4A (n = 6), -75.2 ± 2.1 mV and 8.1 ± 1.0 mV for 1-4B (n = 6), -74.8 ± 1.4 mV and 9.6 ± 0.3 mV for 1-4C (n = 6), -65.8 ± 3.4 mV and 6.9 ± 0.5 mV for 1-4D (n = 4), -61.4 ± 1.6 mV and 9.2 ± 1.7 mV for 1-4E (n = 4), -84.5 ± 1.3 mV and 6.9 ± 0.2 mV for 1-4-1A (n = 9), -64.7 ± 4.8 mV and 11.0 ± 1.2 mV for 1-4-1B (n = 4), and -65.5 ± 2.9 mV and 10.7 ± 0.8 mV for 1-4-1C (n = 4). These factors were measured at 35 °C in slow-activating chimeras (4-1B, 4-1C, 4-1D, 4-1E, 4-1F, 4-1G, 1-4A, 1-4B, and 1-4-1A) and at 25 °C in fast-activating chimeras (4-1A, 1-4 C, 1-4D, 1-4E, 1-4-1B, and 1-4-1C). | ||
The effects of the C-terminal region were further examined in chimeras whose N-termini were derived from HCN1 (from 1-4A to 1-4E in Fig. 2). for 1-4B was increased 3.5-fold compared with for 1C (P < 0.01) although for 1-4C was not statistically different from for 1C (Fig. 2). These results also demonstrate that the introduction of the S6-CNBD linker affected the activation kinetics.
It can be concluded that the S1 domain and the S1-S2 linker mainly determine the difference of activation kinetics between HCN1 and HCN4. This conclusion was further tested by introducing S1 and S1-S2 of HCN4 into 1C (1-4-1A in Fig. 2), which resulted in prolonging 10-fold at -100 mV, whereas introduction of the other two regions did not affect values significantly (1-4-1B, 1-4-1C in Fig. 2).
In all chimeras in Fig. 2, V0.5 values were between -61 and -81 mV, and the slope factors were between 5.6 and 11.0 mV as detailed in the figure legend. We could not find any correlation between the activation time constants and the parameters (V0.5 values and slope factors) of steady-state activation.
Point mutants in S1 and S1-S2 linker did not markedly affect activation time constants
There are only eight amino acid residue differences in S1 and S1-S2 linker between HCN1 and HCN4 (Fig. 3A). Point mutations were introduced into 1C by using site-directed mutagenesis. No single mutation made more than 2-fold longer than 1C wild-type (Fig. 3B). However, 1C-I137T (substitution of 1C Ile137 by Thr), 1C-M141L and 1C-T157K significantly made 1.7-, 1.7- and 1.6-fold longer, respectively. These effects were additive, and double mutations (1C-I137T, M141L) made 4-fold longer. Quadruple mutations (1C-I137T, M141L, T157K, Q159E) made 7.6-fold longer compared with 1C wild-type. This is comparable to that of the chimera 1-4-1A, which made 10-fold longer (Fig. 3B).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 3. Some mutations in S1 and S1-2 linker of HCN1 significantly affected activation time constants during -100 mV hyperpolarizing step A, comparison of sequences for S1 and S1-S2 linker of mouse HCN1 and rabbit HCN4. Identical residues with respect to the mouse HCN1 sequence are represented by dashes. S1 is indicated by a bar below the sequence. B, activation time constants of 1 | ||
The total replacement of S1 and S1-S2 linker changed activation kinetics drastically but was accompanied by a slight shift of voltage dependency
The S1 domain and the S1-S2 linker in 4NC were totally replaced by those of HCN1 (4-1-4A) (Fig. 4A). Activation time constants were measured from the holding potential of -20 mV by step voltage changes made from either -60 or -70 mV to -120 mV in 10 mV steps (Fig. 4D). At less negative potentials, the channel activation was extremely slow. In order to reach steady-state activation during conditioning pulses (e.g. 6 s for HCN4), all experiments in Fig. 4 were performed at 35 °C. Voltage-activation curves were evaluated from the amplitude of tail currents and were fitted with the Boltzmann function (Fig. 4C). The introduction of S1 and S1-S2 of HCN1 into 4NC (4-1-4A) accelerated the activation 3.2-fold at -100 mV (Fig. 4D) and shifted the voltage-activation curve in the depolarizing direction by 5.8 mV (Fig. 4C) compared with 4NC. The additional introduction of the C-terminal region of HCN1 into 4-1-4A (4-1-4-1A) accelerated the activation 9.0-fold at -100 mV compared with 4NC (Fig. 4D). The introduction of S1 and S1-S2 of HCN4 into 1C (1-4-1A) decelerated the activation 16-fold at -100 mV (Fig. 4D) and shifted the voltage-activation curve in the hyperpolarizing direction by 21.1 mV (Fig. 4C) compared with 1C. These results suggest that S1 and S1-S2 linker account for almost half of the differences in activation kinetics between HCN1 and HCN4. The region between S6 and CNBD also accounts for a part of the differences, as shown in Fig. 2 (4-1F vs. 4-1G, 1-4B vs. 1-4C).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 4. Activation and deactivation kinetics and voltage-activation curves at 35 °C in chimeras with reciprocally replaced S1 and S1-S2 linker regions A, schematic representation of HCN1-HCN4 chimeras. The exact points of the crossovers for 1-4-1-4A are shown in parentheses. B, representative current recordings of 1-4-1A and 4-1-4A at 35 °C. The pulse protocol is shown in the top. C, activation curves for 1 | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
HCN channels have a variety of roles, such as rhythmic firing, regulation of dendrite inputs, and presynaptic modulation in the central nervous system (Pape, 1996; Siegelbaum, 2000). Santoro et al. (2000) suggested that the difference in activation kinetics among HCN subtypes might be related to their different physiological roles. However, the structural basis of activation kinetics has remained elusive.
In our previous paper, we described the current activation at -110 mV by the sum of two exponential functions (Ishii et al. 1999), and another group has reported similar results (Santoro et al. 2000). Vaca et al. (2000) and Seifert et al. (1999) fitted the currents with a single exponential function. In our experiments, the currents activated at potentials more positive than -100 mV were well fitted with single exponential functions. However, the currents activated at potentials more negative than -110 mV showed slowly activated components and deviated from single exponential functions. The amplitudes of these slow components were always less than 5 % of those of fast components. Therefore, we adopted a single exponential fit. Some of the differences in activation time course among different research groups might be due to the different expression systems (Xenopus oocytes (Santoro et al. 2000), COS-7 (Ishii et al. 1999), and HEK293 (Seifert et al. 1999; Vaca et al. 2000).
Since Q10 values of activation kinetics measured between 25 and 35 °C were 6.3 and 5.5 for HCN1 and HCN4, respectively, at -100 mV, 6 s step pulses at 35 °C for HCN4 are equivalent to about 60 s pulses at 21 °C, which was used by Seifert et al. (1999). Q10 values are consistent with the data of other groups (Q10 = 4.3 (Tokimasa & Akasu, 1990) and 4.5 (Magee, 1998)). Most of our experiments were performed at 25 °C in order to keep cells alive for a long time (Fig. 2 and Fig. 3). However, some experiments to obtain the voltage dependence of steady-state activation were performed at 35 °C to accelerate the kinetics (Fig. 1 and Fig. 4) because the activation of HCN4 takes an extremely long time particularly at less negative membrane potentials at 25 °C. It would be expected that the change of temperature would not change V0.5 and have little effect on the slope factor (Tsien & Noble, 1969). Actually, the change of temperature between 25 and 35 °C did not affect steady-state voltage activation curves in HCN1 significantly: V0.5 = -64.2 ± 4.4 mV and S = 8.9 ± 1.0 mV (n = 4) at 25 °C, and V0.5 = -63.1 ± 1.0 mV and S = 10.6 ± 0.4 mV (n = 8) at 35 °C.
In HCN channels, it has already been demonstrated that mutations in the N-terminus of S4 affect voltage dependency of activation (Vaca et al. 2000; Chen et al. 2000), but do not significantly affect activation time constants (Vaca et al. 2000). In voltage-gated potassium channels, it has been shown already that the N-terminal domain, S2-S3, S3-S4 linker and S5 are involved in activation kinetics (Pascual et al. 1997; Mathur et al. 1997; Silverman et al. 2000; Gonzalez et al. 2000). However, our results demonstrate that those regions did not markedly affect the activation kinetics in HCN channels. We found two regions affecting activation kinetics, one being S1 and S1-S2, and the other being S6-CNBD. The reciprocal replacements of the whole S1 and S1-S2 region between HCN1 and HCN4 affected the activation kinetics about 16- and 3-fold, respectively (Fig. 4D).
We introduced site-directed mutations in S1 and S1-S2 linker in HCN1 because there are only eight amino acid residues that differ between HCN1 and HCN4 in this region. Each single mutation affected the activation time constant at -100 mV less than 2-fold (Fig. 3). There were three mutations (I137T, M141L and T157K) which significantly affected the activation kinetics, and the effects were 1.6- to 1.7-fold. A different number of charges was introduced only in T157K, with one positive charge gain. I137T introduced a polar but uncharged side chain. M141L is a subtle conservative change. These mutations seem to change the energy barrier for opening the channel although we cannot evaluate how the conformational change was introduced at present.
The effect of S1 and S1-S2, and S6-CNBD seems to be additive (4-1-4-1A in Fig. 4B). These two regions cannot completely explain the whole difference of activation kinetics between HCN1 and HCN4, but they markedly affected the activation kinetics of HCN channels. These results indicate that S1 and S1-S2 linker, and S6-CNBD linker are the crucial components for the activation gating of HCN channels.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| CHEN, J., MITCHESON, J. S., LIN, M. & SANGUINETTI, M. C. (2000). Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel. Journal of Biological Chemistry 275, 36465-36471 | [Abstract/Full Text] |
| DIFRANCESCO, D. (1993). Pacemaker mechanisms in cardiac tissue. Annual Review of Physiology 55, 455-472 | [Medline] |
| GONZALEZ, C., ROSENMAN, E., BEZANILLA, F., ALVAREZ, O. & LATORRE, R. (2000). Modulation of the Shaker K+ channel gating kinetics by the S3-S4 linker. Journal of General Physiology 115, 193-207 | [Abstract/Full Text] |
| ISHII, T. M., TAKANO, M., XIE, L.-H., NOMA, A. & OHMORI, H. (1999). Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. Journal of Biological Chemistry 274, 12835-12839 | [Abstract/Full Text] |
| LUDWIG, A., ZONG, X., JEGLITSCH, M., HOFMANN, F. & 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. & BIEL, M. (1999). Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO Journal 18, 2323-2329 | [Abstract/Full Text] |
| MAGEE, J. C. (1998). Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. Journal of Neuroscience 18, 7613-7624 | [Abstract/Full Text] |
| MATHUR, R., ZHENG, J., YAN, Y. & SIGWORTH, F. J. (1997). Role of the S3-S4 linker in Shaker potassium channel activation. Journal of General Physiology 109, 191-199 | [Abstract/Full Text] |
| MONTEGGIA, L. M., EISCH, A. J., TANG, M. D., KACZMAREK, L. K. & NESTLER, E. J. (2000). Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Molecular Brain Research 81, 129-139 | [Medline] |
| MORONI, A., BARBUTI, A., ALTOMARE, C., VISCOMI, C., MORGAN, J., BARUSCOTTI, M. & DIFRANCESCO, D. (2000). Kinetic and ionic properties of the human HCN2 pacemaker channel. Pflügers Archiv 439, 618-626 | [Medline] |
| PAPE, H.-C. (1996). Queer current and pacemaker: The hyperpolarization activated cation current in neurones. Annual Review of Physiology 58, 299-327 | [Abstract] |
| PASCUAL, J. M., SHIEH, C.-C., KIRSCH, G. E. & BROWN, A. M. (1997). Contribution of the NH2 terminus of Kv2. 1 to channel activation. American Journal of Physiology 273, C1849-1858 | [Medline] |
| SANTORO, B., CHEN, S., LÜTHI, A., PAVLIDIS, P., SHUMYATSKY, G. P., TIBBS, G. R. & SIEGELBAUM, S. A. (2000). Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. Journal of Neuroscience 20, 5264-5275 | [Abstract/Full Text] |
| SANTORO, B., LIU, D. T., YAO, H., BARTSCH, D., KANDEL, E. R., SIEGELBAUM, S. A. & TIBBS, G. R. (1998). Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93, 717-729 | [Medline] |
| SEIFERT, R., SCHOLTEN, A., GAUSS, R., MINCHEVA, A., LICHTER, P. & KAUPP, U. B. (1999). Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proceedings of the National Academy of Sciences of the USA 96, 9391-9396 | [Abstract/Full Text] |
| SIEGELBAUM, S. A. (2000). Presynaptic facilitation by hyperpolarization-activated pacemaker channels. Nature Neuroscience 3, 101-102 | [Medline] |
| SILVERMAN, W. R., TANG, C.-Y., MOCK, A. F., HUH, K.-B. & PAPAZIAN, D. M. (2000). Mg2+ modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3. Journal of General Physiology 116, 663-677 | [Abstract/Full Text] |
| TOKIMASA, T. & AKASU, T. (1990). Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones. Journal of Physiology 420, 409-429 | [Abstract] |
| TSIEN, R. W. & NOBLE, D. (1969). A transition state theory approach to the kinetics of conductance changes in excitable membranes. Journal of Membrane Biology 1, 248-273 | |
| VACA, L., STIEBER, J., ZONG, X., LUDWIG, A., HOFMANN, F. & BIEL, M. (2000). Mutations in the S4 domain of a pacemaker channel alter its voltage dependence. FEBS Letters 479, 35-40 | [Medline] |
| VACCARI, T., MORONI, A., ROCCHI, M., GORZA, L., BIANCHI, M. E., BELTRAME, M. & DIFRANCESCO, D. (1999). The human gene coding for HCN2, a pacemaker channel of the heart. Biochimica et Biophysica Acta 1446, 419-425 | [Medline] |
Acknowledgements
We thank Dr M. Hazama for the generous gift of mouse brain RNA, and M. Fukao and K. Tsuji for technical support. This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T.M.I., M.T. and H.O.) and by a Japan Heart Foundation research grant (to T.M.I.).
Corresponding author
H. Ohmori: Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Email: ohmori{at}nbiol.med.kyoto-u.ac.jp
This article has been cited by other articles:
|
|
 |
|
  S.-W. Ying, F. Jia, S. Y. Abbas, F. Hofmann, A. Ludwig, and P. A. Goldstein Dendritic HCN2 Channels Constrain Glutamate-Driven Excitability in Reticular Thalamic Neurons J. Neurosci., August 8, 2007; 27(32): 8719 - 8732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nof, D. Luria, D. Brass, D. Marek, H. Lahat, H. Reznik-Wolf, E. Pras, N. Dascal, M. Eldar, and M. Glikson Point Mutation in the HCN4 Cardiac Ion Channel Pore Affecting Synthesis, Trafficking, and Functional Expression Is Associated With Familial Asymptomatic Sinus Bradycardia Circulation, July 31, 2007; 116(5): 463 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. V. Vasilyev, Q. Shan, Y. Lee, S. C. Mayer, M. R. Bowlby, B. W. Strassle, E. J. Kaftan, K. E. Rogers, and J. Dunlop Direct Inhibition of Ih by Analgesic Loperamide in Rat DRG Neurons J Neurophysiol, May 1, 2007; 97(5): 3713 - 3721. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. El-Kholy, P. E. MacDonald, J. M. Fox, A. Bhattacharjee, T. Xue, X. Gao, Y. Zhang, J. Stieber, R. A. Li, R. G. Tsushima, et al. Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Pancreatic {beta}-Cells Mol. Endocrinol., March 1, 2007; 21(3): 753 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Ishii, N. Nakashima, and H. Ohmori Tryptophan-scanning mutagenesis in the S1 domain of mammalian HCN channel reveals residues critical for voltage-gated activation J. Physiol., March 1, 2007; 579(2): 291 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chen, J. Wang, L. Zhou, M. S. George, and S. A. Siegelbaum Voltage Sensor Movement and cAMP Binding Allosterically Regulate an Inherently Voltage-independent Closed-Open Transition in HCN Channels J. Gen. Physiol., January 29, 2007; 129(2): 175 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-F. Tse, T. Xue, C.-P. Lau, C.-W. Siu, K. Wang, Q.-Y. Zhang, G. F. Tomaselli, F. G. Akar, and R. A. Li Bioartificial Sinus Node Constructed via In Vivo Gene Transfer of an Engineered Pacemaker HCN Channel Reduces the Dependence on Electronic Pacemaker in a Sick-Sinus Syndrome Model Circulation, September 5, 2006; 114(10): 1000 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Azene, T. Xue, E. Marban, G. F. Tomaselli, and R. A. Li Non-equilibrium behavior of HCN channels: Insights into the role of HCN channels in native and engineered pacemakers Cardiovasc Res, August 1, 2005; 67(2): 263 - 273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-J. Cao and D. Oertel Temperature Affects Voltage-Sensitive Conductances Differentially in Octopus Cells of the Mammalian Cochlear Nucleus J Neurophysiol, July 1, 2005; 94(1): 821 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. Doan, K. Stephans, A. N. Ramirez, P. A. Glazebrook, M. C. Andresen, and D. L. Kunze Differential Distribution and Function of Hyperpolarization-Activated Channels in Sensory Neurons and Mechanosensitive Fibers J. Neurosci., March 31, 2004; 24(13): 3335 - 3343. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-P. Ma, J. Cui, H.-J. Hu, and Z.-H. Pan Mammalian Retinal Bipolar Cells Express Inwardly Rectifying K+ Currents (IKir) With a Different Distribution Than That of Ih J Neurophysiol, November 1, 2003; 90(5): 3479 - 3489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lesso and R. A. Li Helical Secondary Structure of the External S3-S4 Linker of Pacemaker (HCN) Channels Revealed by Site-dependent Perturbations of Activation Phenotype J. Biol. Chem., June 13, 2003; 278(25): 22290 - 22297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Chaplan, H.-Q. Guo, D. H. Lee, L. Luo, C. Liu, C. Kuei, A. A. Velumian, M. P. Butler, S. M. Brown, and A. E. Dubin Neuronal Hyperpolarization-Activated Pacemaker Channels Drive Neuropathic Pain J. Neurosci., February 15, 2003; 23(4): 1169 - 1178. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
