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J. Biol. Chem., Vol. 277, Issue 46, 43588-43592, November 15, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43588    most recent M208477200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, N. Articles by Accili, E. A. Search for Related Content PubMed PubMed Citation Articles by Tran, N. Articles by Accili, E. A. Social Bookmarking                      What's this?

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

Neil Tran, Catherine Proenza, Vincenzo Macri, Fiona Petigara, Erin Sloan, Shannon Samler, and Eric A. Accili^§

From the Ion Channel Laboratory, School of Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

Received for publication, August 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-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 NH₂ termini 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Pacemaker channels are formed by hyperpolarization-activated cyclic nucleotide-gated (HCN)¹ subunits and are important for generating spontaneous activity in a variety of excitable cells (1, 2). 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 (3-5). Co-assembly of different mammalian HCN isoforms has been suggested using electrophysiological analyses (6, 7), and different isoforms have been found in the same cells (8-12). 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 (13). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Yeast Two-hybrid Assays-- 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 and BamHI restriction sites added to the ends of the primers. The PCR fragments were then ligated into pGAD424 and pGBT9 at their EcoRI 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  and  subunits of the skeletal muscle L-type Ca²⁺ channel (14), and were defined as appearance of colonies in >70% of the test transformations. Each interaction was tested in at least six independent transformations.

Mutagenesis and Expression-- The deletion mutants, "HCN2-2-130" and "HCN2-2-154," 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, with BamHI 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 and EcoRI, 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 "HCN2-2-154-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 the BamHI 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.

Electrophysiology-- 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 mM KCl, 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 mM KCl, 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 mM NaCl, 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 M 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,

(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 the p 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.

Immunocytochemistry and Confocal Microscopy-- 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 HCN2-2-154 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.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Conserved Region of the NH₂ Terminus Is a Site of Intersubunit Interaction-- 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 (13).

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Alignment of the NH2 terminus reveals a highly conserved region. The amino acid sequences of the four mammalian HCN channels (mHCN1, -2, -3, and hHCN4) were aligned using ClustalW 1.8 available at The Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). Shading was carried out using Boxshade 3.21 on the Swiss EMBnet node web site (www.ch.embnet.org/software/BOX_form.html). Amino acids highlighted in black represent complete identities, whereas those highlighted in gray represent conserved identities. The vertical line divides the conserved and unconserved regions of the mHCN2 NH2 terminus.

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).

View larger version (65K): [in this window] [in a new window]   Fig. 2.   The site of intersubunit interaction in the NH2 terminus is in the conserved region. cDNA encoding the conserved and unconserved regions of the NH2 terminus of mHCN2 were inserted in-frame into both the binding domain (pGBT9) and activation domain (pGAD424) of the GAL4 transcription factor. The resulting fusion proteins were co-expressed in yeast strain AH 109 and assayed for interaction by activation of a HIS3 reporter gene after incubation of the yeast for 4-7 days on nutritional selection medium (SD/Trp/Leu/His). ID × ID and pGAD × pGBT9 are positive and negative controls, respectively. The positive and negative signs below each colony growth picture indicate positive and negative interactions, respectively, and the numbers in parentheses represent the number of positive or negative interactions over the number of independent yeast transformations performed.

The Conserved Region of the NH₂ Terminus Is Sufficient for Expression of HCN2 Currents-- Previously, we found that the complete removal of the NH₂ terminus of mHCN2 (see construct in Fig. 3D) abolished expression of HCN2 currents and resulted in the localization of channel protein mainly in intracellular compartments (13). Because the conserved domain was found to interact with itself in the yeast two-hybrid assays, we reasoned that this portion of the NH₂ terminus 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 (HCN2-2-130, Fig. 3B). 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 (15), 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 Shaker channels (16).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Schematic representations of the full mHCN2 channel and the three deletion clones. Schematic representations of the wt mHCN2 channel, HCN2-2-130, HCN2-2-154, and HCN2-N showing the six transmembrane domains (S1-S6), the conserved region of the NH2 terminus (gray box), and the predicted coiled-coil domain (striped box). HCN2-2-130 lacks the unconserved region of the NH2 terminus. In HCN2-2-154, the unconserved region and almost half of the conserved region of the NH2 terminus were removed, up to four amino acids from the beginning of the putative coiled-coil region. In HCN2-N, the entire NH2 terminus was removed.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   The 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, HCN2-2-154, HCN2-2-130, or HCN2 in response to voltage steps of 150 mV from a holding potential of 35 mV. Average current densities of Ih (B) and Iinst (C) were recorded from cells expressing green fluorescent protein (n = 9), HCN2 (n = 9), HCN2-2-130 (n = 10), or HCN2-2-154 (n = 11). Single asterisks indicate a significant difference of HCN2 values from the other groups, whereas the double asterisk indicates a significant difference of HCN2-2-130 from all other groups, using an ANOVA followed by Tukey's post-hoc analysis. Confocal images of cells transfected with HCN2-c-Myc (D) or HCN2-2-154-c-Myc (E) are shown. The small arrows point to regions of the plasma membrane conspicuous for the presence or absence of fluorescence. The large arrow (in D) points to a nontransfected cell for comparison.

There are several possible explanations for the reduction in I_h density produced by cells expressing HCN2-2-130. The decrease could reflect a negative shift in the voltage dependence of I_h activation. However, the V_1/2 and k values of I_h activation curves determined from cells expressing HCN2-2-130 were 106.9 ± 4.4 mV and 9.0 ± 0.8 mV, respectively (n = 6). I_h was completely activated at 150 mV. This is similar to what we reported previously for wt mHCN2 (V_1/2 = 101 ± 3.1 mV and k = 12.1 ± 1.7 mV, n = 7 (13, 15)). Thus, the smaller I_h current measured for the HCN2-2-130 at 150 mV was not because of a negative shift in activation curve but probably resulted from a decrease in functional channel number or single channel conductance. Either mechanism is supported by the parallel decreases in I_inst and I_h, but single channel measurements and/or the determination of surface channel number will be required to answer this question.

Another possible explanation for the reduction in current produced by HCN2-2-130 is that the deletion of the unconserved domain could have disrupted the tertiary structure. However, other studies have found that complete exchange of NH₂ termini between different HCN mammalian isoforms (with unrelated unconserved regions) did not significantly alter I_h gating properties (17, 18). Together with the relatively normal voltage dependence of HCN2-2-130 channels, these data suggest that the unconserved region of the NH₂ terminus does not greatly influence the proper assembly and folding of HCN channels. Another explanation could be that the unconserved domain contains primary sequences that specifically promote assembly and/or expression (16, 19).

A Putative Coiled Coil Domain within the Conserved Region Is Not Sufficient for Expression of HCN2 Currents or Trafficking of Protein to the Plasma Membrane-- An artificial coiled coil oligomerization sequence, inserted in place of the conserved NH₂-terminal tetramerization or "T1" domain of Shaker K⁺ channels, has recently been shown to retain efficient expression of these channels (16, 20). We searched the mHCN2 NH₂ terminus for regions with high probabilities of forming coiled coils, using a statistical coiled coil prediction program.² A relatively polar region of 14 amino acids (from Gln¹⁵⁹ to Ala¹⁷²) with a high probability (83.9%) of forming a coiled-coil configuration was identified within the conserved domain of the NH₂ terminus. Based on this finding, we constructed a deletion mutant, HCN2-2-154, further removing the amino acids up to this putative coiled-coil region (Fig. 3C) and then determined whether functional expression was retained. Cells transfected with the HCN2-2-154 clone were pulsed to 150 mV from a holding potential of 35 mV for 1 s. We found that this deletion abolished I_h and reduced I_inst to the same level as that of control cells transfected with only green fluorescent protein (Fig. 4, A-C).

Two possible explanations for the inability of the HCN2-2-154 mutant to produce currents are that: (a) the mutant channels were assembled and exported to the cell surface, but channel function was eliminated by the deletion; or (b) the mutant channels were expressed at reduced levels on the plasma membrane. To discriminate between these two possibilities, we examined the subcellular localization of HCN2-2-154 compared with that of the wt HCN2. To do this, we inserted the c-Myc epitope on the COOH-terminal end of both wt HCN2 and HCN2-2-154. Chinese hamster ovary cells expressing HCN2-c-Myc had immunofluorescent labeling evident on the cell surface (Fig. 4D). In contrast, the HCN2-2-154-c-Myc protein was predominantly localized in the perinuclear regions of the cell (e.g. Golgi, endoplasmic reticulum, or degredatory compartments), with little or no apparent surface immunofluorescence (Fig. 4E). Given the interactions of the conserved domain in the yeast two-hybrid assays and the elimination of currents in the HCN2-2-154 mutant, the retention of the HCN2-2-154 mutant protein in intracellular compartments most likely represents inefficiently assembled channels and reduced trafficking of channels to the plasma membrane as has been proposed for Shaker channels lacking conserved regions of the NH₂ terminus (16).

Summary and Perspectives-- We have shown that a region of 52 amino acids of the NH₂ terminus, which is highly conserved among mammalian isoforms of HCN channels, is important for NH₂-terminal interactions, plasma membrane localization, and expression of pacemaker currents. This region may play a role in isoform co-assembly that is similar to the role of the T1 domains in Shaker channels. Although Shaker channels can form in the absence of the NH₂ terminus (21), T1 domains may support tetramerization by bringing subunits into close proximity at the beginning of assembly in the endoplasmic reticulum (22) and increase the effective local concentration of compatible subunits (16). Formation of functional Shaker channels is retained when the T1 domain is replaced by a coiled coil peptide (GCN4-LI), an oligomerization domain structurally unrelated to the T1 domains of Shaker channels, thus indicating different types of structures that may serve to facilitate subunit assembly (20, 16). It will be interesting to find out whether the conserved NH₂ terminus of HCN channels is structurally, as well as functionally, similar to the corresponding regions of Shaker channels.

	ACKNOWLEDGEMENTS

We thank Damiano Angoli and Kristin Zahynacz (from our laboratory) for assistance in some experiments, and Andreas Ludwig (Technische Universität Munchen) for the mouse HCN2 clone.

	FOOTNOTES

^* This work was supported by grants from the Heart and Stroke Foundation of Canada, the Heart and Stroke Foundation of British Columbia and The Yukon (to E. A. A. and C. P.), the Canadian Institutes for Health Research, and the American Health Assistance Foundation (to E. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115.

^§ To whom correspondence should be addressed. Tel.: 604-291-4574; Fax: 604-291-3040; E-mail: eaaccili@sfu.ca.

Published, JBC Papers in Press, August 21, 2002, DOI 10.1074/jbc.M208477200

² The program was created by Lupas et al. (24) and was based on the prediction protocol proposed by David Parry (26). It can be found on the Swiss EMBnet node web site at www.ch.embnet.org/software/COILS_form.html.

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/46/43588    most recent M208477200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tran, N. Articles by Accili, E. A. Search for Related Content PubMed PubMed Citation Articles by Tran, N. Articles by Accili, E. A. Social Bookmarking                      What's this?