Molecular Characterization of the Hyperpolarization-activated Cation Channel in Rabbit Heart Sinoatrial Node

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (I for I h) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brainI f cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1–3. The transmembrane region of HAC4 was most homologous to partially cloned mouse I f BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at −87.2 ± 2.8 mV under control conditions and at −64.4 ± 2.6 mV in the presence of intracellular 0.3 mm cAMP. The reversal potential was −34.2 ± 0.9 mV in 140 mm Na+oand 5 mm K+o versus 10 mm Na+i and 145 mmK+i. These results indicate that HAC4 formsI f in rabbit heart SA node. We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (I for I h) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brainI f cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1–3. The transmembrane region of HAC4 was most homologous to partially cloned mouse I f BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at −87.2 ± 2.8 mV under control conditions and at −64.4 ± 2.6 mV in the presence of intracellular 0.3 mm cAMP. The reversal potential was −34.2 ± 0.9 mV in 140 mm Na+oand 5 mm K+o versus 10 mm Na+i and 145 mmK+i. These results indicate that HAC4 formsI f in rabbit heart SA node. The hyperpolarization-activated cation channel (I f) 1The abbreviations used are: I f, hyperpolarization-activated cation channel(s); SA, sinoatrialwas first described in rabbit heart sinoatrial (SA) node (1Yanagihara K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; 385: 11-19Crossref PubMed Scopus (202) Google Scholar, 2DiFrancesco D. Ojeda C. J. Physiol. (Lond.). 1980; 308: 353-367Crossref Scopus (119) Google Scholar). TheI f current was characterized by activation by hyperpolarizing voltage steps; mixed permeability for Na+and K+; inhibition by extracellular Cs+, not by Ba2+; and a positive shift in the voltage-dependent activation curve by intracellular cyclic nucleotide (1Yanagihara K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; 385: 11-19Crossref PubMed Scopus (202) Google Scholar, 2DiFrancesco D. Ojeda C. J. Physiol. (Lond.). 1980; 308: 353-367Crossref Scopus (119) Google Scholar, 3DiFrancesco D. Ferroni A. Mazzanti M. Trobba C. J. Phyiol. (Lond.). 1986; 377: 61-88Crossref PubMed Scopus (357) Google Scholar, 4DiFrancesco D. Tortora P. Nature. 1991; 351: 145-147Crossref PubMed Scopus (649) Google Scholar, 5Frace M. Maruoka M. Noma A. Pfluegers Arch. Eur. J. Physiol. 1992; 421: 97-99Crossref PubMed Scopus (12) Google Scholar, 6Ho W.-K. Brown H.F. Noble D. Pfluegers Arch. Eur. J. Physiol. 1994; 426: 68-74Crossref PubMed Scopus (27) Google Scholar). Based on the above physiological properties, the functional roles ofI f in the SA node have been discussed in many publications. I f is one of the inward currents that generate pacemaker depolarization (7Denyer J.C. Brown H.F. J. Physiol. (Lond.). 1990; 429: 401-409Crossref Scopus (128) Google Scholar, 8Ginneken A.G.C. Giles W. J. Physiol. (Lond.). 1991; 434: 57-83Crossref Scopus (106) Google Scholar, 9DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (673) Google Scholar). The pacemaker cells of the SA node are coupled to surrounding atrial myocytes through gap junctions. Since atrial myocytes have more negative resting membrane potentials, they hyperpolarize pacemaker cells electrotonically (10Watanabe E. Honjo H. Anno T. Boyett M.R. Kodama I. Toyama J. Am. J. Physiol. 1995; 269: H1742-H1753Google Scholar). Pacemaker cell I f is activated under this condition; therefore; the inward I f current is likely to limit the level of hyperpolarization of pacemaker cells (1Yanagihara K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; 385: 11-19Crossref PubMed Scopus (202) Google Scholar). Recently, three full-length (mouse BCNG-1, -2, and -4; corresponding to HAC2, -1, and -3, respectively) and one partial (mouse BCNG-3) mammalian cDNA clones encoding I f were isolated from a mouse brain cDNA library (11Santoro B. Grant S.G.N. Bartsch D. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14815-14820Crossref PubMed Scopus (228) Google Scholar, 12Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 13Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 14Clapham D.E. Neuron. 1998; 21: 5-7Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and one cDNA clone was isolated from sea urchin testis (SPIH) (15Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (379) Google Scholar). However, despite the physiological significance of I f in the SA node, its molecular characteristics still remain unclear. Therefore, we have screened a rabbit heart SA node cDNA library and isolated a cDNA (HAC4). HAC4 is composed of 1150 amino acid residues and most likely encodes I f. In this study, we demonstrate the amino acid sequence of HAC4, the distribution of HAC4 mRNA in cardiac tissue, and the electrophysiological properties of HAC4 heterologously expressed in COS-7 cells. The cloning procedure was performed as described previously (16Ishii T. Moriyoshi K. Sugihara H. Sakurada K. Kadotani H. Yokoi M. Akazawa C. Shigemoto R. Mizuno N. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 2836-2843Abstract Full Text PDF PubMed Google Scholar). Briefly, cDNA templated by mRNA isolated from rat brain was used as a DNA template for polymerase chain reaction amplification. The 5′ (P1) and 3′ (P2) sequences were derived from mouse HAC2 (13Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar) and are as follows (N represents A/G/C/T): P1, 5′-ATG(C/T)TNTG(T/C)AT(T/C/A)GGNTA(T/C)GG-3′; and P2, 5′-AT(A/G)TA(A/G)TCNCCNGG(T/C)TG(A/G)AA-3′. Polymerase chain reaction amplification was performed according to the following schedule: five cycles at 94 °C for 1 min, 46 °C for 1 min, and 72 °C for 2 min, followed by 26 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. The polymerase chain reaction products were electrophoresed on a 1% agarose gel, excised, and purified with QIAEX-II (QIAGEN Inc.) for subsequent subcloning and sequence determination. Through this procedure, we identified a clone for a rat homologue of mouse HAC2, pR1. A cDNA library was prepared from rabbit heart SA node regions. Using pR1 as a probe, 5 × 104 phage clones of the cDNA library were hybridized for the isolation of a new clone (washed with 2× SSC at 55 °C). Five positive clones were isolated and sequenced. All of them were identical. A representative clone, pIH1, was subjected to sequence determination. Both strands of the cDNA sequence were determined by the chain termination method (BigDye Terminator Cycle Sequencing, Applied Biosystems, Inc.). The clone contains 3396 base pairs of cDNA that comprises a large open reading frame. However, the cDNA does not possess a termination codon in the C-terminal region. Therefore, we performed 3′-rapid amplification of cDNA ends (Marathon cDNA amplification kit, CLONTECH) and obtained the C-terminal region of the clone by using proofreadingTaq polymerase (LA Taq, TaKaRa). We added the C-terminal region amplified by the proofreading Taq polymerase to pIH1 by using a SacI restriction site. Northern blots, prepared with 2 μg of poly(A)+ mRNA isolated from the indicated rabbit tissues (atrium does not contain the SA node region), were probed in hybridization solution (Life Technologies, Inc.) and 50% formamide at 42 °C with a radiolabeled DNA fragment derived from the coding region of pIH1 (corresponding to amino acids 707–1116), washed with 0.1× SSC and 0.1% SDS at 65 °C, and exposed to x-ray film at −80 °C with an intensifying screen for 84 h. HAC4 cDNA and green fluorescent protein S65A cDNA (a gift from Dr. K. Moriyoshi) were subcloned into independent PCI vectors (Promega), and the mixture of vectors were transfected into COS-7 cells using LipofectAMINE (Life Technologies, Inc.) following the manufacturer's instructions. The vector amounts were 1.6 μg/35-mm dish for HAC4 and 0.4 μg/35-mm dish for green fluorescent protein. COS-7 cells (Riken) were cultured on coverslips in Dulbecco's modified essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.) and antibiotics. 36–48 h after transfection, a coverslip was transferred to the recording chamber on an inverted microscope (TMD300, Nikon), and patch-clamp experiments were carried out in green fluorescent protein-positive cells using Axopatch 200B amplifier (Axon Instruments, Inc.). The data were directly recorded on the hard disk of an IBM-PC compatible computer thorough an AD converter (Digipack 1200, Axon Instruments, Inc.) and were analyzed using commercially available software (pClamp6 and Clampex7, Axon Instruments, Inc.). The data points represent the means ± S.E. The statistical difference was evaluated using Student's unpaired t test. The composition of the bathing solution was 140 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES. The pH was adjusted to 7.4 with NaOH. Appropriate amounts of KCl, CsCl, and BaCl2 were added in some experiments. The pipette solution contained 130 mmKCl, 5 mm HEPES, 5 mm EGTA, 5 mmMgATP, and 5 mm disodium creatine phosphate. The pH was adjusted to 7.4 with KOH. The final K+ concentration was ∼145 mm. In some experiments, 0.3 mm cAMP was added to the pipette solution, and the pH was readjusted to 7.4 with KOH. All patch-clamp experiments were carried out at 35 °C by perfusing the bathing solution through a water jacket. The transmembrane region of rat HAC2 was amplified by the polymerase chain reaction method with a rat brain cDNA mixture and was used to screen a rabbit SA node cDNA library. The most 5′ methionine codon in the positively hybridizing clone (pIH1) initiated an open reading frame that did not contain a termination codon in the 3′-terminal region. 3′-Rapid amplification of cDNA ends was performed to obtain the 3′-terminal region, and pIH1 lacked 103 base pairs of the 3′-coding region. The 3′-terminal region was amplified by proofreading Taq polymerase and attached to pIH1. The sequence (HAC4) (Fig. 1) predicts a protein of 1150 amino acid residues with six transmembrane domains, a pore region, and a cyclic nucleotide-binding domain. HAC4 shows 85–90 and 80–96% identities to HAC1–3 in the transmembrane region and the cyclic nucleotide-binding domain, respectively. HAC4 is most related to mouse BCNG-3. Although full-length mouse BCNG-3 cDNA has not been cloned, the partial sequence of mouse BCNG-3 was 96% homologous to the transmembrane region of HAC4. The predicted N- and C-terminal regions of HAC4 are notably longer than those of the rest of the HAC clones. The last five amino acid residues (corresponding to positions 1146–1150) at the C terminus were conserved in all HAC clones. The amino acid sequence corresponding to positions 1045–1055 was also conserved in all HAC clones. The rest of the C-terminal regions were remarkably diverse. To examine the expression of HAC4 mRNA, we performed Northern blotting (Fig.2). Since the C-terminal regions are diverse among HAC family members, we synthesized a radiolabeled DNA fragment derived from the coding region corresponding to amino acids 707–1116. It is clear from Fig. 2 that HAC4 was the most highly expressed in the SA node among the cardiac tissues examined. Unlike HAC1–3, HAC4 was not significantly expressed in brain. The size of the mRNA for HAC4 was estimated to be 7.1 kilobases, although presumably a partially processed transcript was detected. Fig.3 A shows representative current traces of HAC4 expressed in COS-7 cells. When a voltage step was more negative than −60 mV, slowly activating inward currents were activated. Such a current was not observed when COS-7 cells were transfected with an empty vector (data not shown). The current activation appeared to be the sum of two exponential time courses. For example, time constants of current activation were 384 ± 71 and 2275 ± 319 ms at −110 mV (n = 6). These values were similar to those reported for the I fcurrent of rabbit SA node cells (17Maruoka M. Nakashima Y. Takano M. Ono K. Noma A. J. Physiol. (Lond.). 1994; 477: 423-435Crossref Scopus (38) Google Scholar). Fig. 3 B shows the current-voltage (I-V) relationship. The closed circles indicate the amplitude of the initial current measured at the beginning of hyperpolarizing pulses. The open circlesare the steady-state I-V relationship measured at the end of pulses. It is clear from Fig. 3 B that the threshold of current activation was between −60 and −70 mV. The voltage dependence of current activation was further analyzed by of currents (Fig. 3 control hyperpolarizing voltage steps to and mV did not the When the voltage step was −60 mV, a current was The amplitude of the current as the voltage more negative and at mV. It is that intracellular cAMP a positive shift in the voltage dependence of the activation of the I f and currents in a The was obtained at mm cAMP D. Tortora P. Nature. 1991; 351: 145-147Crossref PubMed Scopus (649) Google Scholar, 13Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar). In with an current was by hyperpolarization to mV in the presence of intracellular 0.3 mm cAMP In Fig. 3 the current (I measured at mV was by the (I in the presence and of 0.3 mm cAMP. In we the to the data = is the h is the membrane potential for half-maximal and is the control h was ± 2.8 mV, was ± mV (n = In the presence of 0.3 mm the of the positive ± 2.6 mV (n = did not significantly ± 0.4 mV (n = The shift h was mV, was than those in mammalian clones as mouse (corresponding to 2 B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar) (13Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar). The I fcurrent of the SA node and HAC clones is by extracellular and D. Ferroni A. Mazzanti M. Trobba C. J. Phyiol. (Lond.). 1986; 377: 61-88Crossref PubMed Scopus (357) Google Scholar, J.C. Brown H.F. J. Physiol. (Lond.). 1990; 429: 401-409Crossref Scopus (128) Google Scholar, 12Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 13Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (379) Google M. Nakashima Y. Takano M. Ono K. Noma A. J. Physiol. (Lond.). 1994; 477: 423-435Crossref Scopus (38) Google Scholar, K. Maruoka F. Noma A. Pfluegers Arch. Eur. J. Physiol. 1994; PubMed Scopus Google Scholar). We examined the of and on HAC4 currents as in A. The of the was estimated by the amplitude of currents in the presence and of mV, 3 mm partially the HAC4 The of the was ± was than reported for mouse and The current was also by extracellular 3 ± of the HAC4 It is that and K+ f in pacemaker cells D. Ferroni A. Mazzanti M. Trobba C. J. Phyiol. (Lond.). 1986; 377: 61-88Crossref PubMed Scopus (357) Google Scholar, 5Frace M. Maruoka M. Noma A. Pfluegers Arch. Eur. J. Physiol. 1992; 421: 97-99Crossref PubMed Scopus (12) Google Scholar, 6Ho W.-K. Brown H.F. Noble D. Pfluegers Arch. Eur. J. Physiol. 1994; 426: 68-74Crossref PubMed Scopus (27) Google Scholar). In we examined the of HAC4 by the reversal was determined by between curve of initial currents in the I-V and the I-V curve of currents (data not were −34.2 ± 0.9 mV (n = in 5 and ± 0.9 mV (n = in mm The permeability for and was from the following = is the permeability for is the permeability for K+; is the reversal and and are the were ± in 5 mm K+o and ± in mm It is also clear from Fig. B that extracellular the inward in a In the of extracellular current was activated the These properties are in with those of the I fcurrent in pacemaker cells M. Maruoka M. Noma A. Pfluegers Arch. Eur. J. Physiol. 1992; 421: 97-99Crossref PubMed Scopus (12) Google Scholar). and also I f (17Maruoka M. Nakashima Y. Takano M. Ono K. Noma A. J. Physiol. (Lond.). 1994; 477: 423-435Crossref Scopus (38) Google Scholar). In Fig. we the permeability of and through HAC4. When 140 was with 140 in the presence of 5 the amplitude of the current the reversal potential to ± mV (n = When we estimated by intracellular was The time of the of the current in 140 mm In 140 mm the reversal potential was ± mV (n = These properties were similar to those reported for I f in pacemaker cells (17Maruoka M. Nakashima Y. Takano M. Ono K. Noma A. J. Physiol. (Lond.). 1994; 477: 423-435Crossref Scopus (38) Google Scholar). In this study, we have cloned a cDNA (HAC4) and that HAC4 encodes I f. HAC4 is composed of 1150 amino acid Both the cytoplasmic N- and C-terminal regions of HAC4 are longer than those of HAC1–3. We not the that the terminus of HAC4 was not we have not obtained a clone that has an codon in of the However, the region contained and showed homology with HAC clones. the cDNA of HAC4 functional I f currents when expressed in COS-7 cells. These the that the cDNA of HAC4 contains the coding The C-terminal region of HAC4 showed remarkably low homology with HAC clones. The amino acid sequence of the transmembrane region of HAC4 was homologous to (mouse to HAC2 (mouse to a of mouse and to mouse BCNG-3 D.E. Neuron. 1998; 21: 5-7Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The partial sequence of the region amino acid of mouse BCNG-3 was also homologous to HAC4 Therefore, HAC4 be the rabbit homologue of mouse BCNG-3. It is to this a partial sequence of mouse BCNG-3 has been determined amino and in the amino acid sequence of the C-terminal region of mouse BCNG-3 has not been Although HAC4 and mouse BCNG-3 are highly expression in BCNG-3 is expressed in cardiac tissue, not in the SA node. BCNG-3 is also expressed in mouse brain and B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). In HAC4 mRNA is highly expressed in rabbit heart SA node. In of cardiac and HAC4 are not We not have for this In Northern we used the sequence that to amino acids 707–1116. the amino acid sequence of the corresponding region of mouse BCNG-3 not been we not the that the C-terminal region was not homologous between HAC4 and mouse BCNG-3. be that the expression is to the difference of In with the distribution of HAC4 mRNA, the electrophysiological properties of HAC4 those of f current reported in pacemaker cells isolated from rabbit SA node. In pacemaker the ofI f and currents was the activation of I f was by the of I (1Yanagihara K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; 385: 11-19Crossref PubMed Scopus (202) Google Scholar, K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; PubMed Scopus Google Scholar). In this study, was current in COS-7 cells than the heterologously expressed HAC4 to voltage-dependent ofI f more The threshold of current activation was between −60 and −70 mV under control conditions and between and mV in the presence of intracellular 0.3 mm cAMP. The h of HAC4 was more positive than that of mouse In HAC4, the positive shift in h was mV in the presence of 0.3 mm was than that in mV by mm mouse mV by 3 mm In pacemaker the activation curve of I f was from cell to cell D. Ferroni A. Mazzanti M. Trobba C. J. Phyiol. (Lond.). 1986; 377: 61-88Crossref PubMed Scopus (357) Google Scholar, 8Ginneken A.G.C. Giles W. J. Physiol. (Lond.). 1991; 434: 57-83Crossref Scopus (106) Google Scholar). be in two cAMP concentration be from cell to I f in the SA node be a composed of HAC and composition be from cell to although we not have on the expression level of HAC clones in rabbit SA node. In the to that the possess a between those of HAC4 and HAC family The functional of I f in the SA node is to generate pacemaker depolarization (7Denyer J.C. Brown H.F. J. Physiol. (Lond.). 1990; 429: 401-409Crossref Scopus (128) Google Scholar, 8Ginneken A.G.C. Giles W. J. Physiol. (Lond.). 1991; 434: 57-83Crossref Scopus (106) Google Scholar, 9DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (673) Google Scholar) and to limit the hyperpolarization of pacemaker cells by with atrial myocytes (1Yanagihara K. Irisawa H. Pfluegers Arch. Eur. J. Physiol. 1980; 385: 11-19Crossref PubMed Scopus (202) Google Scholar, E. Honjo H. Anno T. Boyett M.R. Kodama I. Toyama J. Am. J. Physiol. 1995; 269: H1742-H1753Google Scholar). The significance of I the of pacemaker potential still to be a of (17Maruoka M. Nakashima Y. Takano M. Ono K. Noma A. J. Physiol. (Lond.). 1994; 477: 423-435Crossref Scopus (38) Google Scholar, K. Noma A. Irisawa H. J. Physiol. 1980; PubMed Scopus Google Scholar, A. M. Irisawa H. Pfluegers Arch. Eur. J. Physiol. PubMed Scopus Google Scholar, T. R. W. Pfluegers Arch. Eur. J. Physiol. PubMed Scopus Google Scholar, J. Ono K. Noma A. J. Physiol. (Lond.). 1995; Scopus Google Scholar). of the HAC a to this To an is essential to the molecular characteristics ofI f in the SA node. In this study, we have that HAC4 I f in the SA node. molecular of HAC4 in the SA node the of the physiological ofI f. We are to Dr. K. Ono for reading We M. and K. for