A Conserved Domain in the NH2 Terminus Important for Assembly and Functional Expression of Pacemaker Channels

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclicnucleotide-gated (HCN) subunits. Previously, we suggested that the NH2 termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH2 terminus of the HCN2 subunit that is responsible for interactions between NH2termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH2-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH2-terminal determinant of subunit co-assembly and trafficking of pacemaker channels. Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclicnucleotide-gated (HCN) subunits. Previously, we suggested that the NH2 termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH2 terminus of the HCN2 subunit that is responsible for interactions between NH2termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH2-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH2-terminal determinant of subunit co-assembly and trafficking of pacemaker channels. Pacemaker channels are formed byhyperpolarization-activated cyclicnucleotide-gated (HCN) 1The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-gated channel; I h, hyperpolarization-activated current; I inst, instantaneous current; PBS, phosphate-buffered saline; ANOVA, analysis of variance; wt, wild type subunits and are important for generating spontaneous activity in a variety of excitable cells (1Accili E.A. Proenza C. Baruscotti M. DiFrancesco D. News Physiol. Sci. 2002; 17: 32-37PubMed Google Scholar, 2Kaupp U.B. Seifert R. Annu. Rev. Physiol. 2001; 63: 235-257Crossref PubMed Scopus (306) Google Scholar). Their primary amino acid sequence predicts a structure similar to those of voltage-gated potassium channels and cyclic nucleotide-gated channels. Thus, HCN subunits are thought to have six transmembrane helices with cytoplasmic amino and carboxyl termini, and to co-assemble as tetramers when forming functional channels. Four mammalian HCN isoforms (HCN1–4) are known (3Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 29: 717-729Abstract Full Text Full Text PDF Scopus (583) Google Scholar, 4Ludwig A. Zong X. Jeglitsch M. Hoffman F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 5Ishii T.M. Takano M. Xie L.H. Noma A. Ohmori H. J. Biol. Chem. 1999; 274: 12835-12839Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Co-assembly of different mammalian HCN isoforms has been suggested using electrophysiological analyses (6Chen S. Wang J. Siegelbaum S.A. J. Gen. Phys. 2001; 117: 491-504Crossref PubMed Scopus (328) Google Scholar, 7Ulens C. Tytgat J. J. Biol. Chem. 2001; 276: 6069-6072Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and different isoforms have been found in the same cells (8Santoro B. Chen C. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar, 9Moosmang S. Stieber J. Zong X. Biel M. Hofmann F. Ludwig A. Eur. J. Biochem. 2001; 268: 1646-1652Crossref PubMed Scopus (354) Google Scholar, 10Moroni A. Gorza L. Beltrame M. Gravante B. Vaccari T. Bianchi M.E. Altomare C. Longhi R. Heurteaux C. Vitadello M. Malgaroli A. DiFrancesco D. J. Biol. Chem. 2001; 276: 29233-29241Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11Monteggia L.M. Eisch A.J. Tang M.D. Kaczmarek L.K. Nestler E.J. Brain Res. Mol. Brain Res. 2000; 81: 129-139Crossref PubMed Scopus (183) Google Scholar, 12Stevens D.R. Seifert R. Bufe B. Muller F. Kremmer E. Gauss R. Meyerhof W. Kaupp U.B. Lindemann B. Nature. 2001; 413: 631-635Crossref PubMed Scopus (189) Google Scholar). These findings suggest that the formation of heteromeric channels contributes to the diversity of pacemaker current phenotypes described in vivo. Recently, we suggested that NH2-terminal interactions are required for subunit co-assembly and targeting of functional channels to the plasma membrane (13Proenza C. Tran N. Angoli D. Zahynacz K. Balcar P. Accili E.A. J. Biol. Chem. 2002; 277: 29634-29642Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, we have not yet determined the region(s) responsible. To identify the critical region, we subdivided the NH2 terminus based on homology among the different mammalian isoforms of HCN channels. An alignment of NH2-terminal amino acid sequences from these isoforms revealed a 52-amino acid domain, which has a high sequence identity (>90%), and is located immediately adjacent to the first putative transmembrane domain (S1). Using yeast two-hybrid assays, confocal imaging, and patch clamp electrophysiology, we have found that the conserved domain interacted with itself and was required for plasma membrane localization of channel protein and expression of HCN2 currents. Our data suggests that the conserved domain of the NH2 terminus is important for subunit co-assembly and trafficking of pacemaker channels. The coding sequences for the unconserved domain (residues 1–130) and conserved domain (residues 131–182) of the mHCN2 NH2 terminus were each inserted in-frame into both the GAL4 activation- and binding-domain plasmids, pGAD424 and pGBT9 (Clontech, Palo Alto, CA). The two regions were amplified using PCR, with EcoRI andBamHI restriction sites added to the ends of the primers. The PCR fragments were then ligated into pGAD424 and pGBT9 at theirEcoRI and BamHI restriction sites. The resulting constructs were confirmed by automated DNA sequencing (Centre for Molecular Medicine and Therapeutics, University of British Columbia, Canada). The constructs were assayed for interaction by expression in the yeast strain AH109. Pooled yeast colonies expressing the fusion proteins were collected from plates containing synthetic medium lacking tryptophan and leucine (SD/−Trp/−Leu) and were then spread onto test plates containing medium lacking tryptophan, leucine, and histidine (SD/−Trp/−Leu/−His). The test plates were examined for the appearance of colonies after incubation for 3–7 days at 30 °C. Positive interactions were compared with a control interaction consisting of the interaction domains from the α and β subunits of the skeletal muscle L-type Ca2+ channel (14Proenza C. Wilkens C. Lorenzon N.M. Beam K.G. J. Biol. Chem. 2000; 275: 23169-23174Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and were defined as appearance of colonies in >70% of the test transformations. Each interaction was tested in at least six independent transformations. The deletion mutants, “HCN2-Δ2–130” and “HCN2-Δ2–154,” were constructed by replacing an EcoRI-AccI restriction fragment of the wild type channel with a PCR product lacking the coding sequence for residues 2–130 and 2–154, respectively. For the c-Myc tagged channel, “HCN2-c-Myc,” the sequence encoding residues 1–863 of the wt mHCN2 channel was first amplified using PCR, withBamHI and EcoRI restriction sites added to the ends of the primers. The PCR product was then inserted into a mammalian c-Myc expression vector, pcDNA3.1/myc-His (Invitrogen), using the common restriction sites BamHI andEcoRI, such that the c-Myc protein is expressed on the COOH-terminal end of the resulting fusion protein. The same method was also used to construct “HCN2-Δ2–154-c-Myc,” except that the coding sequence for residues 155–863 was amplified in the PCR amplification step instead, and an ATG start codon was added after theBamHI restriction site. All constructs were confirmed by automated DNA sequencing (Centre for Molecular Medicine and Therapeutics, University of British Columbia). Chinese hamster ovary-K1 cells (American Type Culture Collection, Manassas, VA) were maintained in Hams' F-12 medium supplemented with antibiotics and 10% fetal bovine serum, and incubated at 37 °C with 5% CO2. Cells were plated onto glass coverslips in 35-mm dishes. One day after plating, mammalian expression vectors encoding wild type (wt) or mutant mHCN channels (2 μg/dish) were transiently co-transfected into the cells along with the green fluorescent protein reporter plasmid (0.3 μg/dish) using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cells expressing the transfected DNA were identified by the appearance of green fluorescence 24–48 h after transfection. One to 2 days following transfection, a shard of coverslip plated with cells was transferred to a recording chamber (∼200 μl volume) and continually perfused (0.5–1.0 ml/min) with a low K+ extracellular solution (5.4 mmKCl, 135 mm NaCl, 0.5 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, pH 7.4, with NaOH). Following rupture of the patch membrane, the solution was changed to a high K+ recording solution (135 mmKCl, 5.4 mm NaCl, 0.5 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, pH 7.4, with KOH) to maximize current amplitude. The patch pipettes were filled with a solution containing 130 mm K-aspartate, 10 mmNaCl, 0.5 mm MgCl2, 5 mm HEPES, and 1 mm EGTA and adjusted to pH 7.4 with KOH. Whole cell currents were measured using borosilicate glass electrodes, which had a resistance of 2.0–4.0 mΩ when filled with the intracellular solution. Currents were recorded using an Axopatch 200B amplifier and Clampex software (Axon Instruments). Data were filtered at 2 kHz and were analyzed using Clampfit (Axon Instruments) and Origin (Microcal) software. All experiments were conducted at room temperature (20–22 °C). Currents were not leak-subtracted. Instantaneous currents were taken as the peak current measured immediately after the capacitive transient. The voltage dependence of activation was determined from tail currents at −65 mV following 2-s test pulses ranging from 60 to −150 mV, in 30-mV steps. Normalized tail current amplitudes were plotted as a function of test potential and values were fit with a Boltzmann function, f(V)=Imax/(1+e(V1/2−V)/k)(Eq. 1) to determine the midpoint of activation (V 1/2) and slope factor (k). Single test pulses were often followed by a 200–500-ms pulse to +5 mV to ensure complete channel deactivation, and the resting current was always allowed to return to its baseline value before subsequent voltage pulses. Statistical comparisons were performed using an ANOVA followed by Tukey's post-hoc analysis; significance was assumed if thep value was <0.05. Data are reported as mean ± S.E., and n values represent the number of cells measured, which were from a minimum of three separate transfections for each value reported. One day after transfection, cells on coverslips were washed with phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde in PBS for 5 min. The cells were washed with PBS (2× for 10 min), permeabilized using 0.2% Triton X-100, and blocked with 10% normal goat serum for 10 min. After one wash with PBS containing 1% normal goat serum, cells were incubated with a mouse monoclonal antibody (Invitrogen) specific to the c-Myc epitope present on the COOH termini of the wild type and HCN2-Δ2–154 mHCN2 constructs at a dilution of 1:3200 in PBS with 1% normal goat serum for 2 h at room temperature. The antibody was removed, cells were again washed with PBS (3× for 5 min), and then incubated with a goat anti-mouse secondary antibody tagged with Cy3 (Jackson Laboratories, West Grove, PA) at a dilution of 1:600 in PBS with 1% normal goat serum for 1 h at room temperature in the dark. After washing in PBS (3× for 5 min), coverslips were mounted on slides using Permount (Fisher). Cells were examined using confocal microscopy (Zeiss LSM 5 Pascal), and images were taken using a ×63 oil immersion objective lens at an excitation wavelength of 535 nm. An alignment of amino acid sequences of the amino termini of four mammalian HCN isoforms (mHCN1, mHCN2, mHCN3, and hHCN4) revealed a region of high sequence identity (>90%) (Fig.1). This region is 52 amino acid residues long (Ser131-Asp182) and is located immediately adjacent to the first putative transmembrane domain (S1). We hypothesized that this conserved region could mediate the interactions between complete NH2 termini observed previously in our lab (13Proenza C. Tran N. Angoli D. Zahynacz K. Balcar P. Accili E.A. J. Biol. Chem. 2002; 277: 29634-29642Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Using yeast two-hybrid assays, we tested the conserved and unconserved domains of HCN2 for self-interactions. The conserved (Ser131-Asp182) and unconserved (Met1-Gly130) domains were expressed in the yeast strain AH 109 as fusion proteins with the binding domain and activation domain of the GAL4 transcription factor. The presence of yeast colonies on SD/−Trp/−Leu/−His nutritional selection medium was used as the indicator of interactions between test proteins. Yeast expressing the conserved domain exhibited robust growth, similar to that of yeast expressing the positive control interaction domains of the skeletal muscle L-type-calcium channel. In contrast, no growth was observed for yeast expressing either the unconserved domain of the NH2 terminus or the negative control, which consisted of the GAL4 activation and binding domains alone (Fig.2). The conserved and unconserved domain constructs were also expressed with the empty vectors (pGAD and pGBT9) to ensure that the positive results were not because of cross-reactivity with the GAL4 portions of the fusion proteins. The results of those control tests were negative (n = 6 transformations each, data no shown). Previously, we found that the complete removal of the NH2 terminus of mHCN2 (see construct in Fig. 3 D) abolished expression of HCN2 currents and resulted in the localization of channel protein mainly in intracellular compartments (13Proenza C. Tran N. Angoli D. Zahynacz K. Balcar P. Accili E.A. J. Biol. Chem. 2002; 277: 29634-29642Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Because the conserved domain was found to interact with itself in the yeast two-hybrid assays, we reasoned that this portion of the NH2terminus would promote channel assembly and trafficking, and therefore restore the expression of HCN2 currents. To test this possibility, we constructed a deletion mutant containing only the conserved domain (HCN2-Δ2–130, Fig. 3 B). When expressed in Chinese hamster ovary cells, this construct produced both the slowly activating current (I h) and the instantaneous current (I inst) that are characteristic of mHCN2 channels (15Proenza C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), although both currents were significantly smaller than those recorded from cells expressing wt mHCN2 (Fig.4, A–C). Thus, the conserved domain of the NH2 terminus of HCN2 was sufficient to rescue functional expression, probably by promoting channel assembly and trafficking to the plasma membrane as has been suggested for the conserved NH2-terminal domains of Shakerchannels (16Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar).Figure 4The conserved region is sufficient for functional expression, but the putative coiled-coil region is not. A, current traces recorded from cells expressing green fluorescent protein, HCN2-Δ2–154, HCN2-Δ2–130, or HCN2 in response to voltage steps of −150 mV from a holding potential of −35 mV. Average current densities of I h (B) and I inst (C) were recorded from cells expressing green fluorescent protein (n = 9), HCN2 (n = 9), HCN2-Δ2–130 (n = 10), or HCN2-Δ2–154 (n = 11). Single asterisksindicate a significant difference of HCN2 values from the the a significant difference of HCN2-Δ2–130 from using an ANOVA followed by Tukey's post-hoc images of cells transfected with or are The to regions of the plasma membrane for the presence or of The D) to a cell for are for the h produced by cells expressing The could a negative in the voltage dependence of I h However, and values h activation determined from cells expressing HCN2-Δ2–130 were ± mV and ± mV, (n = I h was at −150 mV. This is similar to we reported previously for wt mHCN2 (V = ± mV and = ± mV, n = (13Proenza C. Tran N. Angoli D. Zahynacz K. Balcar P. Accili E.A. J. Biol. Chem. 2002; 277: 29634-29642Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, C. Angoli D. Agranovich E. Macri V. Accili E.A. J. Biol. Chem. 2002; 277: 5101-5109Abstract Full Text Full Text PDF PubMed Scopus (91) Google Thus, the smaller I h current measured for the HCN2-Δ2–130 at −150 mV was not because of a negative in activation but probably resulted from a in functional channel number or channel is by the inst and I h, but channel the of channel number required to this for the in current produced by HCN2-Δ2–130 is that the deletion of the unconserved domain could have the However, have found that complete of NH2 termini between different HCN mammalian isoforms unconserved not significantly I h C. Altomare C. A. E. Baruscotti M. A. DiFrancesco D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Chen S. Siegelbaum S.A. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar). with the relatively normal voltage dependence of HCN2-Δ2–130 these data suggest that the unconserved region of the NH2 terminus not the assembly and of HCN channels. could that the unconserved domain primary sequences that specifically promote assembly expression (16Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar, D. N. M. Jan Y.N. Jan L.Y. 2001; PubMed Scopus Google Scholar). An inserted in of the conserved NH2-terminal or domain of has been to expression of these channels (16Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar, A. Jan Y.N. Jan L.Y. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We the mHCN2 NH2 terminus for regions with high of forming using a was by A. J. PubMed Scopus Google and was based on the by PubMed Scopus Google Scholar). found on the at relatively polar region of amino to with a high of forming a coiled-coil was identified within the conserved domain of the NH2 on this we constructed a deletion HCN2-Δ2–154, further the amino to this putative coiled-coil region 3 and then determined functional expression was Cells transfected with the HCN2-Δ2–154 were to −150 mV from a holding potential of −35 mV for 1 We found that this deletion abolished I h and inst to the same as that of control cells transfected with only green fluorescent protein for the of the HCN2-Δ2–154 mutant to currents are the mutant channels were and to the cell but channel function was eliminated by the or the mutant channels were expressed at on the plasma membrane. To between these two we examined the localization of HCN2-Δ2–154 compared with that of the wt To we inserted the c-Myc epitope on the COOH-terminal end of both wt HCN2 and Chinese hamster ovary cells expressing had on the cell In contrast, the protein was predominantly localized in the perinuclear regions of the cell or with or no the interactions of the conserved domain in the yeast two-hybrid and the of currents in the HCN2-Δ2–154 the of the HCN2-Δ2–154 mutant protein in intracellular compartments channels and trafficking of channels to the plasma membrane as has been channels lacking conserved regions of the NH2 terminus (16Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar). We have that a region of 52 amino of the NH2 which is highly conserved among mammalian isoforms of HCN is important for NH2-terminal plasma membrane and expression of pacemaker currents. This region a in isoform co-assembly that is similar to the of the domains channels. channels in the of the NH2 terminus C. Biol. 1999; PubMed Scopus Google Scholar), domains by subunits into at the of assembly in the J. D. C. 2001; PubMed Scopus Google and the of subunits (16Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar). of functional channels is when the domain is by a an domain to the domains different of that to subunit assembly A. Jan Y.N. Jan L.Y. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar). to the conserved NH2terminus of HCN channels is as as similar to the regions of channels. We Angoli and Zahynacz our for in and Ludwig for the mouse HCN2

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