Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2001 Nov 15;537(Pt 1):93–100. doi: 10.1111/j.1469-7793.2001.0093k.x

Determinants of activation kinetics in mammalian hyperpolarization-activated cation channels

Takahiro M Ishii 1, Makoto Takano 1, Harunori Ohmori 1
PMCID: PMC2278938  PMID: 11711564

Abstract

  1. The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS-7).

  2. The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at −100 mV.

  3. In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1–S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics.

  4. The S1 domain and S1–S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1–S2 region of HCN1 by that of HCN4 resulted in a 10− to 20-fold slowing.

  5. The results indicate that S1 and S1–S2, and S6–CNBD are the crucial components for the activation gating of HCN channels.

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

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 1ΔC and to the N and C-termini-deleted HCN4 as 4ΔNΔC (Fig. 1A). 1ΔC lacks 289 amino acid residues at the C-terminus in mouse HCN1. 4ΔNΔC 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:Inline graphic

Figure 1. Activation kinetics and voltage-activation curves of HCN1, HCN4 and their deletion mutants.

Figure 1

Open in a new tab

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 (1ΔC and 4ΔNΔC) at 35 °C. The pulse protocol is shown at the top. C, the currents obtained from −100 mV voltage pulse (holding potential = −20 mV) were fitted to single exponential functions to obtain the activation time constants in HCN1 and HCN4 at 35 °C. The dashed lines show the fits and the continuous lines the currents. D, activation time constants as a function of voltage during hyperpolarizing steps for HCN1 (n = 6), 1ΔC (n = 6), HCN4 (n = 9) and 4ΔNΔC (n = 5) at 35 °C. E, activation curves for HCN1, 1ΔC, HCN4 and 4ΔNΔC at 35 °C. The continuous lines represent fits of relative tail current amplitudes to Boltzmann functions. The V0.5 and slope factors were −63.1 ± 1.0 mV and 10.6 ± 0.4 mV for HCN1 (n = 8), −63.4 ± 1.1 mV and 9.7 ± 0.4 mV for 1ΔC (n = 8), −62.5 ± 1.4 mV and 5.7 ± 0.2 mV for HCN4 (n = 12), and −72.6 ± 1.4 mV and 5.3 ± 0.3 mV for 4ΔNΔC (n = 5), respectively.

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 MΩ when measured in experimental solutions. The series resistance was 5–20 MΩ 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((VmV0.5)/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.

Figure 4. Activation and deactivation kinetics and voltage-activation curves at 35 °C in chimeras with reciprocally replaced S1 and S1–S2 linker regions.

Figure 4

Open in a new tab

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 1ΔC, 4ΔNΔC, 1–4-1A and 4–1-4A at 35 °C. The continuous lines represent fits of relative tail current amplitudes to Boltzmann functions. The V0.5 and slope factors, respectively, were −63.4 ± 1.1 mV and 9.7 ± 0.4 mV for 1ΔC (n = 8), −72.6 ± 1.4 mV and 5.3 ± 0.3 mV for 4ΔNΔC (n = 5), −84.5 ± 1.3 mV and 6.9 ± 0.2 mV for 1–4-1A (n = 9), and −66.8 ± 1.8 mV and 7.0 ± 0.1 mV for 4–1-4A (n = 11). D, activation time constants as a function of voltage during hyperpolarizing steps for 1ΔC (n = 6), 4ΔNΔC (n = 5), 1–4-1A (n = 8), 4–1-4A (n = 5) and 4–1-4-1A (n = 8) at 35 °C.

RESULTS

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 (τ) 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 τ 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 (1ΔC) 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 (4ΔNΔC) 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 1ΔC and 4ΔNΔC (Fig. 2A). τ 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 4ΔNΔC are shown (from 4–1A to 4–1G). As the region derived from HCN4 was longer, τ became longer. Especially, the introduction of the S1 region from HCN4 markedly prolonged τ (between 4–1A and 4–1B: 6.5-fold; P < 0.01). Besides, τ values between 4–1B and 4–1C were significantly different (1.7-fold; P < 0.01). τ 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.

Figure 2. Comparison of activation time constants among HCN1-HCN4 chimeras during −100 mV hyperpolarizing voltage steps.

Figure 2

Open in a new tab

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). τ for 1–4B was increased 3.5-fold compared with τ for 1ΔC (P < 0.01) although τ for 1–4C was not statistically different from τ for 1ΔC (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 1ΔC (1-4-1A in Fig. 2), which resulted in prolonging τ 10-fold at −100 mV, whereas introduction of the other two regions did not affect τ 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 1ΔC by using site-directed mutagenesis. No single mutation made τ more than 2-fold longer than 1ΔC wild-type (Fig. 3B). However, 1ΔC-I137T (substitution of 1ΔC Ile137 by Thr), 1ΔC-M141L and 1ΔC-T157K significantly made τ 1.7-, 1.7 and 1.6-fold longer, respectively. These effects were additive, and double mutations (1ΔC-I137T, M141L) made τ 4-fold longer. Quadruple mutations (1ΔC-I137T, M141L, T157K, Q159E) made τ 7.6-fold longer compared with 1ΔC wild-type. This τ is comparable to that of the chimera 1–4-1A, which made τ 10-fold longer (Fig. 3B).

Figure 3. Some mutations in S1 and S1-2 linker of HCN1 significantly affected activation time constants during −100 mV hyperpolarizing step.

Figure 3

Open in a new tab

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 1ΔC mutants during a −100 mV hyperpolarization step at 25 °C. * Significantly different from 1ΔC, P < 0.05. The number of experiments in each group is given in parentheses.

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 4ΔNΔC 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 4ΔNΔC (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 4ΔNΔC. 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 4ΔNΔC (Fig. 4D). The introduction of S1 and S1–S2 of HCN4 into 1ΔC (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 1ΔC. 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).

DISCUSSION

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.

Acknowledgments

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.).

References

  1. Chen J, Mitcheson JS, Lin M, Sanguinetti MC. Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel. Journal of Biological Chemistry. 2000;275:36465–36471. doi: 10.1074/jbc.M007034200. [DOI] [PubMed] [Google Scholar]
  2. DiFrancesco D. Pacemaker mechanisms in cardiac tissue. Annual Review of Physiology. 1993;55:455–472. doi: 10.1146/annurev.ph.55.030193.002323. [DOI] [PubMed] [Google Scholar]
  3. Gonzalez C, Rosenman E, Bezanilla F, Alvarez O, Latorre R. Modulation of the Shaker K+ channel gating kinetics by the S3-S4 linker. Journal of General Physiology. 2000;115:193–207. doi: 10.1085/jgp.115.2.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ishii TM, Takano M, Xie L-H, Noma A, Ohmori H. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. Journal of Biological Chemistry. 1999;274:12835–12839. doi: 10.1074/jbc.274.18.12835. [DOI] [PubMed] [Google Scholar]
  5. Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature. 1998;393:587–591. doi: 10.1038/31255. [DOI] [PubMed] [Google Scholar]
  6. Ludwig A, Zong X, Stieber J, Hullin R, Hofmann F, Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO Journal. 1999;18:2323–2329. doi: 10.1093/emboj/18.9.2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Magee JC. Dendritic hyperpolarization-activated currents modify the integrative properties of hippocampal CA1 pyramidal neurons. Journal of Neuroscience. 1998;18:7613–7624. doi: 10.1523/JNEUROSCI.18-19-07613.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mathur R, Zheng J, Yan Y, Sigworth FJ. Role of the S3-S4 linker in Shaker potassium channel activation. Journal of General Physiology. 1997;109:191–199. doi: 10.1085/jgp.109.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Monteggia LM, Eisch AJ, Tang MD, Kaczmarek LK, Nestler EJ. Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain. Molecular Brain Research. 2000;81:129–139. doi: 10.1016/s0169-328x(00)00155-8. [DOI] [PubMed] [Google Scholar]
  10. Moroni A, Barbuti A, Altomare C, Viscomi C, Morgan J, Baruscotti M, DiFrancesco D. Kinetic and ionic properties of the human HCN2 pacemaker channel. Pflügers Archiv. 2000;439:618–626. doi: 10.1007/s004249900225. [DOI] [PubMed] [Google Scholar]
  11. Pape H-C. Queer current and pacemaker: The hyperpolarization activated cation current in neurones. Annual Review of Physiology. 1996;58:299–327. doi: 10.1146/annurev.ph.58.030196.001503. [DOI] [PubMed] [Google Scholar]
  12. Pascual JM, Shieh C-C, Kirsch GE, Brown AM. Contribution of the NH2 terminus of Kv2. 1 to channel activation. American Journal of Physiology. 1997;273:C1849–1858. doi: 10.1152/ajpcell.1997.273.6.C1849. [DOI] [PubMed] [Google Scholar]
  13. Santoro B, Chen S, Lüthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, Siegelbaum SA. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. Journal of Neuroscience. 2000;20:5264–5275. doi: 10.1523/JNEUROSCI.20-14-05264.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell. 1998;93:717–729. doi: 10.1016/s0092-8674(00)81434-8. [DOI] [PubMed] [Google Scholar]
  15. Seifert R, Scholten A, Gauss R, Mincheva A, Lichter P, Kaupp UB. 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. 1999;96:9391–9396. doi: 10.1073/pnas.96.16.9391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Siegelbaum SA. Presynaptic facilitation by hyperpolarization-activated pacemaker channels. Nature Neuroscience. 2000;3:101–102. doi: 10.1038/72038. [DOI] [PubMed] [Google Scholar]
  17. Silverman WR, Tang C-Y, Mock AF, Huh K-B, Papazian DM. Mg2+ modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3. Journal of General Physiology. 2000;116:663–677. doi: 10.1085/jgp.116.5.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tokimasa T, Akasu T. Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones. Journal of Physiology. 1990;420:409–429. doi: 10.1113/jphysiol.1990.sp017920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tsien RW, Noble D. A transition state theory approach to the kinetics of conductance changes in excitable membranes. Journal of Membrane Biology. 1969;1:248–273. doi: 10.1007/BF01869785. [DOI] [PubMed] [Google Scholar]
  20. Vaca L, Stieber J, Zong X, Ludwig A, Hofmann F, Biel M. Mutations in the S4 domain of a pacemaker channel alter its voltage dependence. FEBS Letters. 2000;479:35–40. doi: 10.1016/s0014-5793(00)01837-8. [DOI] [PubMed] [Google Scholar]
  21. Vaccari T, Moroni A, Rocchi M, Gorza L, Bianchi ME, Beltrame M, DiFrancesco D. The human gene coding for HCN2, a pacemaker channel of the heart. Biochimica et Biophysica Acta. 1999;1446:419–425. doi: 10.1016/s0167-4781(99)00092-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES