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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada
Submitted 5 February 2008 ; accepted in final form 3 July 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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pacemaker channel function; protein export; trafficking; hyperpolarization-activated cyclic nucleotide-gated channel
In HCN2 channels, the cyclic nucleotide-binding domain (CNBD), located in the COOH terminus, appears to be an important determinant of cell surface expression, in addition to its better known role as regulator of channel opening (31), based on two studies (1, 20). First, complex glycosylation is abolished in HCN2 mutants lacking the CNBD, which supports its necessity for export of the channel from the ER (1). Second, we identified a subdomain of the CNBD that strongly promotes cell surface and functional expression; mutants lacking this subdomain do not generate current and are retained intracellularly (20). This same subdomain, which consists of the A and B helices and the interceding β-barrel, exerts tonic inhibition of channel opening in response to hyperpolarization (5). Whether the complete subdomain is required for both functions, perhaps by conferring a shared conformational change, or includes distinct regions that contribute to each function is not known.
The mechanism by which the CNBD subdomain promotes cell surface expression is poorly understood. To date, it has been shown that HCN2 channels lacking the entire CNBD do not appear to form functional channels when expressed in mammalian cells (20, 25), but do in Xenopus laevis oocytes (31). Moreover, in mammalian cells these same mutant channels form homotetramers that do not exit the ER (1) but can be rescued by coassembly with wild-type HCN1 subunits to form functional, heteromeric channels (20). Together, these data suggest that the CNBD promotes ER export and cell surface expression in at least two ways. First, the efficiency with which channels correctly fold, assemble, and move through quality control pathways could be enhanced. Second, a step in forward trafficking may be augmented.
Studies by our group and by Akhavan et al. (1) have narrowed the region of the HCN2 CNBD subdomain that promotes cell surface expression to the B-helix (1, 20). We showed that a COOH-terminal truncation mutant that lacks the B-helix eliminates functional expression and thus has the same phenotype as the complete CNBD truncation mutant, whereas that which retains the B-helix produces wild-type levels of current (20). Akhavan et al. (1) showed that the proline at the end of the B-helix is required for complex glycosylation, which is presumably indicative of cell surface expression. Whether the B-helix contributes to inhibition of channel opening is not known and remains to be investigated.
The hypothesis that determinants within the B-helix of the HCN2 channel play a role in both cell surface expression and inhibition of hyperpolarization-activated opening forms the basis of the current study. Herein, we identify a four-amino acid motif (EEYP) that promotes ER export and cell surface expression of functional HCN2 channels by augmenting a step in forward trafficking and/or enhancing the efficiency of folding and assembly. However, the EEYP motif does not contribute to the inhibition of channel opening, indicating that cell surface expression and inhibition of channel opening are carried out, at least in part, by separate regions of the CNBD subdomain.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CNBD (525) and HCN2-EEYP-C-term (615). Other CNBD mutants constructed were as follows: HCN2-4A, generated by replacing EEYP (616–619) with four alanines using overlapping mutagenic PCR; HCN2-4A-C-term, which has a stop codon inserted immediately after the alanine string (620) in the HCN2-4A construct; and HCN2-EEAA, HCN2-AAYP, and HCN2-EEYM [identified as P619M by Akhavan et al. (1)] by overlapping mutagenic PCR.
NH2-terminal deletion mutants, some of which were characterized in previous studies (20, 26), were used comparatively with the COOH-terminal mutants. These included HCN2-2-130, HCN2-2-137, HCN2-2-138, HCN2-2-143, HCN2-2-154, and HCN2-2-182, which were constructed by replacing an EcoRI-AccI restriction fragment of the wild-type HCN2 channel with a PCR product lacking the coding sequence for residues 2-130, 2-137, 2-138, 2-143, 2-154, and 2-182, respectively. All constructs were cloned into the pcDNA3.1 mammalian expression vector (Invitrogen, Burlington, ON, Canada).
A glycosylation-deficient HCN2 mutant (HCN2-N380Q) was also created by replacing Asn380 with Gln using overlapping mutagenic PCR. In addition, COOH-terminal-myc (c-myc)-tagged versions of wild-type HCN2 and the NH2-terminal deletion mutant channels were constructed using PCR, with BamHI and EcoRI common restriction sites incorporated on the ends of the primers, to allow subcloning into pcDNA 3.1-myc such that the myc protein is expressed on the COOH-terminal end of the resulting fusion protein.
Lastly, HCN2 channels were constructed with an extracellular hemagglutinin (HA) tag between the third and fourth transmembrane segments (S3-S4). An HCN2 construct with the HA epitope in pBluescript (a kind gift from Michael Sanguinetti, University of Utah) was excised from pBluescript and ligated into pcDNA3.1 for expression in Chinese hamster ovary (CHO) cells (HCN2-HA). The HA epitope was also inserted into the correlate domain of HCN2-CNBD, HCN2-2-182, HCN2-EEYP-C-term, HCN2-4A-C-term, and HCN2-4A, which were each then cloned into pcDNA3.1 using the same restriction enzyme sites.
All constructs were confirmed by automated DNA sequencing (Nucleic Acid Protein Services, University of British Columbia). The HA- and c-myc-tagged wild-type channels were found to be functional using whole cell patch-clamp electrophysiology (see below).
CHO-K1 cells (American Tissue Type Culture Collection, Manassas, VA) were maintained in Ham's F-12 medium supplemented with penicillin, streptomycin, and 10% fetal bovine serum and were 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 or mutant HCN2 channels (2 µg/dish) were transiently cotransfected into the cells along with the green fluorescent protein reporter plasmid (0.5–0.7 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.
Whole cell patch-clamp electrophysiology.
One to two days following transfection, a shard of coverslip plated with cells was transferred to a recording chamber (
200 µl vol) 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, 1.9 mM CaCl2, and 5 mM HEPES, adjusted to pH 7.4 with NaOH). Following rupture of the patch membrane, this was switched to a high K+ extracellular solution (135 mM KCl, 5.4 mM NaCl, 0.5 mM MgCl2, 1.9 mM CaCl2, and 5 mM HEPES, adjusted to pH 7.4 with KOH) to maximize current amplitude. The patch pipettes were filled with a solution containing 130 mM potassium 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 (Sutter, Novato, CA), 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). Series resistance was not compensated, and currents were not leak-subtracted. The voltage dependence of activation was determined from tail currents at –65 mV following 2–3 s test pulses; interpulse intervals were 10–15 s and were followed by a 200–500 ms pulse to +5 mV to ensure complete channel deactivation. The resting current was always at its baseline value before subsequent voltage (V) pulses.
Normalized tail current amplitudes were plotted as a function of test potential, and values were fitted with a Boltzmann function
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Immunocytochemistry and microscopy. For these experiments, HCN2 constructs containing an HA-epitope inserted between the third and fourth transmembrane segments were used to identify relative cell surface expression. Two to three days after transfection, cells on coverslips were washed with phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde in PBS for 5 min. Thereafter, they were washed with PBS, were either permeabilized using 0.2% Triton X-100 or left unpermeabilized, and were then blocked with 10% normal goat serum (NGS). After one wash with PBS containing 1% NGS, cells were incubated with a mouse monoclonal antibody specific to the HA-epitope (Sigma, Oakville, ON) at a dilution of 1:500 in PBS with 1% NGS for 1 h at room temperature. The antibody-containing solution was removed, cells were washed with PBS/NGS 1%, and then incubated with a goat anti-mouse secondary antibody tagged with Alexa 488 (Invitrogen) at a dilution of 1:1,500 in PBS with 1% NGS for 1 h at room temperature in the dark. This solution was removed, and then, after being washed with PBS/NGS 1%, the coverslips were mounted on slides using Gelmount (Sigma) and sealed with clear nail polish. Cells were examined using structured illumination (Zeiss Apotome Imager Z1) with a 63x oil immersion objective lens at wavelengths specific for the Alexa 488 fluorescent protein tag.
Western blot analysis.
Each sample subjected to Western blot analysis was derived from cells on 35-mm plates that had been lysed in 100 µl of lysis buffer containing 50 mM Tris at pH 8.0, 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 2 mM each of Na3VO4 and NaF, and 10 µg/ml each of aprotinin, pepstatin, and leupeptin. Samples were left on ice for 30 min, during which time they were vortexed every 5 min for 5 s. After centrifugation to remove cell debris (25,000 g, 25 min), protein concentration of the supernatant was determined by Bradford assay. Samples of supernatant (20 µg) were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (8%) and electroblotted to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Mississauga, ON, Canada), unless otherwise indicated. Blots were washed three times in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) and then blocked with 5% nonfat dry milk (Bio-Rad) in TBST for 1 h at room temperature. Blots were then incubated with one of the following antibodies: 1) a rabbit polyclonal antibody specific to the COOH terminus of HCN2 (Affinity Bioreagents, Golden, CO); 2) a rabbit polyclonal antibody specific to the NH2 terminus of HCN2 (Alomone Laboratories, Jerusalem, Israel); or 3) a mouse monoclonal antibody (Invitrogen) specific to the myc epitope—at dilutions of 1:500, 1:200, and 1:3,000 in TBST with 5% nonfat dry milk, respectively, for 2.5 h at room temperature. Blots were washed in TBST for 10 min, three times, and then incubated with horseradish peroxidase-conjugated to either goat anti-rabbit or goat anti-mouse IgG, accordingly, at 1:3,000 dilution in 5% nonfat dry milk with TBST for 1 h at room temperature; they were subsequently washed three times in TBST. Signals were obtained with ECL Western Blotting Detection Reagents (GE Healthcare, Baie d'Urfe, QC, Canada). Protein loading was controlled by probing all Western blots with either goat anti-GAPDH antibody or rabbit anti-ACTIN (both from Santa Cruz Biotechnology, Santa Cruz, CA).
In some experiments, densitometry was carried out to determine the intensities of bands of interest in the Western blots, using ImageJ software (http://rsb.info.nih.gov/ij/). A rectangle of fixed size was centered on the band of interest. Within each designated region the density was determined and corrected for background.
Proteinase K treatment. Twenty-four hours after transfection, cells were washed in ice-cold PBS three times and incubated with or without 20 µg/ml proteinase K (PK) (BioShop, McGill University, Montreal, QC, Canada) in PK buffer (10 mM HEPES, 150 mM NaCl, and 2 mM CaCl2) at 37°C for 30 min. To stop the PK reaction, a blocking buffer (25 mM EDTA and 20 mM PMSF) was added to all samples for 10 min at 4°C. Cells were harvested by centrifugation at 5,000 rpm for 5 min at 4°C and were then washed with ice-cold PBS twice. Pellets were resuspended in lysis buffer and processed for Western blotting as described above.
Sucrose gradient analysis. Cell lysates of HCN2 and HCN2-4A were subjected to high-speed centrifugation at 140,000 g for 45 min at 4°C. The supernatants (volume chosen to obtain 200 µg protein following Bradford assay) were layered on top of a 5–40% nondenaturing continuous sucrose gradient, made with lysis buffer. Molecular mass protein standards, including bovine serum albumin (66 kDa), alcohol dehydrogenase (151 kDa), and thyroglobulin (669 kDa), 200 µg each, were each layered on separate sucrose gradients. Samples were centrifuged for 16 h at 106,000 g at 4°C, and 13 equal volume fractions were collected serially from the bottom of the gradient. Subsequently, each set of fractions was subjected to Western blotting as described above, with the following changes. Proteins were transferred onto Immobilon-FL PVDF membranes (Millipore, Bedford, MA). Following incubation with the rabbit polyclonal antibody specific for the HCN2 COOH terminus, the blots were washed in TBST and then labeled for 1 h with a secondary anti-rabbit antibody conjugated to Alexa Fluor 680 (1:40,000) (Molecular Probes, Eugene, OR) for analysis on a LI-COR Odyssey imager (Lincoln, NE). For each blot, pixel intensity of each fraction was determined by densitometry and normalized to the highest intensity.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-helices (Fig. 1). An eight-stranded β-barrel in the center of the CNBD forms a basket in which cAMP binds and is shielded from solvent and phosphodiesterases. The most conserved feature among the CNBDs is contained within this barrel—the phosphate binding cassette (PBC), composed of β-strand 6, a short helix, and β-strand 7. A buried arginine that binds to the exocyclic phosphate of cAMP and a glutamate that binds the ribose 2'-OH are conserved features of the PBC.
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and RIβ, all from mouse, shows both similarities and differences among them (Fig. 1). The similarities are greatest in two regions. First, as discussed, regions corresponding to the PBC contain key conserved residues. Second, are regions between β-strand 1 and 4, which may be important for shielding cAMP from solvent and from phosphodiesterases. Sequence alignments, as well as more sophisticated structural alignments, of protein kinases and HCN2 show that the similarities in the CNBD fall off in the middle of the B-helix (3). Our alignment, which includes CNGA3 and ERG1 ion channel sequences, revealed a conserved four-amino acid motif in the distal B-helix among the channels that was not found in the protein kinases. This motif corresponds to EEYP in HCN2, TEYP in CNGA3, and DMYP in ERG1. The presence of this conserved hydrophilic motif in these ion channels but not in the protein kinases raises the possibility that it may confer the channel-specific functions of inhibition of channel opening and cell surface expression attributed to the CNBD. Support for the role of EEYP in channel cell surface expression, in particular, comes from a study in which mouse HCN2 channels possessing a methionine in place of the proline (P619M) reduces complex glycosylation (1); however, the effects of this mutation on folding, assembly, and channel function was not assessed.
The EEYP motif promotes ER export and cell surface expression of mature HCN2.
To investigate whether the EEYP motif promotes the expression of functional channels, we constructed a series of HCN2 COOH-terminal deletion and substitution mutants that target this region (Fig. 2). The deletion mutants were HCN2-EEYP-C-term (the EEYP motif and downstream COOH terminus is deleted, eliminating residues 616-863), HCN2-4A-C-term (the EEYP motif is replaced by four alanines and the remainder of the COOH terminus is deleted), and finally, HCN2-4A (full-length HCN2 with four alanines in place of the EEYP motif).
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136 kDa (mature) and 114 kDa (immature), whereas only the immature form was identified in cells expressing HCN2-N380Q, as expected (16). We found that the mature band is consistently heavier by 22 kDa. In HCN2-4A, the same sequence length as wild-type HCN2, a band at 114 kDa alone was identified. In cells expressing HCN2-EEYP-C-term and HCN2-4A-C-term, two bands at 90 and 83 kDa were identified. Importantly, a band corresponding to a mature form of EEYP mutant channels was not seen in any of the Western blots (12/12 transfections), whereas a mature form of the wild-type channel was present in blots from the same transfections. Because the 22-kDa increase in molecular mass seen upon complex glycosylation of HCN2 was not seen in the three EEYP mutant channels, these likely remain in a simple glycosylated and/or unglycosylated state. Thus, the EEYP motif promotes complex glycosylation of HCN2 channels and thus ER export. (Fig. 3A).
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To link directly EEYP-mediated changes in complex glycosylation, and thus ER export, to cell surface localization, PK experiments were carried out on intact cells transfected with HCN2, HCN2-4A, or HCN2-N380Q, and complex glycosylation was examined (Fig. 3C). Exposure of intact cells to PK would be expected to cleave extracellular regions of only those channels localized to the plasma membrane. As shown, exposure to PK resulted in the elimination of the mature complex glycosylated form of the wild-type channel and produced an additional lower-molecular-mass broad band indicative of cleaved by-products. Exposure of cells expressing HCN2-4A or HCN2-N380Q to PK did not produce any additional bands. Notably, there was no effect of PK on the immature (114 kDa) form of these channels. These data not only show that the mature complex glycosylated form of the channel is present on the cell surface, as was appreciated by Ahkavan et al. (1), but, specifically, that the EEYP motif promotes this pattern of cell surface expression.
The EEYP motif is not required to form functional channels and does not contribute to inhibition of channel opening.
The reduction in mature protein for the mutant EEYP channels (above) suggests that they are retained in the ER, but it does not provide information about the ability of the mutant channels to fold, assemble, and function. Are the mutant channels irreversibly misfolded? The parallel observation that small amounts of EEYP-lacking protein traffic to the cell surface (Fig. 3B) suggested that the EEYP motif is not required to form channels that reach the plasma membrane. To test whether these channels reach the plasma membrane in functional form, the densities of hyperpolarization-activated current (If) were determined in transiently transfected CHO cells using whole cell patch-clamp electrophysiology. If densities produced by HCN2-EEYP-C-term, HCN2-4A-C-term, and HCN2-4A were significantly reduced compared with those of wild-type HCN2 channels. This is illustrated in Fig. 4A, where representative current traces elicited by a 2-s hyperpolarizing pulse to –150 mV (at which the channels were at, or close to, full activation) from a holding potential of –35 mV are shown. If densities at –150 mV are shown in the bar graph in Fig. 4B. These data are consistent with the notion that the EEYP motif promotes export of channels from the ER to the plasma membrane and, importantly, is not required to form functional channels. Thus, a small number of functional EEYP mutant channels are able to make it to the plasma membrane, as foreshadowed by Fig. 3B. The lack of the mature forms of the EEYP mutants by Western blot (Fig. 3A) suggests that the levels of mature protein were below detection or that the immature forms contribute to the observed current. The latter explanation seems less likely given that the immature bands are not modified by PK (Fig. 3C). Removal of the EEYP motif could also reduce channel activity, which would contribute to lower levels of If.
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Next, we wanted to determine whether the EEYP motif contributes to inhibition of channel opening by hyperpolarization. A limiting factor was that the EEYP mutant channels produced levels of If that were often too small to analyze. This issue could be circumvented by selection of relatively large CHO cells that produced adequate levels of If to permit accurate analysis. We thus repeated our measurements of If in large CHO cells transfected with HCN2-EEYP-C-term, HCN2-4A-C-term, and HCN2-4A (Fig. 5A). To determine whether the EEYP contributes to basal inhibition of channel opening, If activation curves were generated from the ratio of tail current amplitudes elicited at the –65 mV voltage step (Fig. 5A, arrows; 5B). If the EEYP contributes to the inhibition of channel opening by the CNBD, then we would expect the activation curves to be shifted in the positive direction in channels lacking this motif. However, we found that the activation curves for all three EEYP mutants were not statistically different from wild type. The similarity in channel activation curves indicates that the EEYP does not contribute to basal inhibition of channel opening by the CNBD.
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The EEYP motif regulates cell surface expression by a mechanism that does not lead to substantive degradation or disruption of subunit assembly. To this point, the data suggest that there are at least two ways in which the EEYP may regulate ER export and cell surface expression of functional HCN2 channels. First, the efficiency with which the channel folds, assembles, and moves through the trafficking pathway may be facilitated by the EEYP motif and, conversely, compromised by its absence. In this scenario, the majority of EEYP mutant channels would be identified as misfolded. Under some circumstances, and depending on the cellular checkpoint at which the channels are identified, the channels would be subject to rapid degradation (29). Second, the EEYP motif may promote specifically the export of correctly folded channels from the ER to the cell surface. In this case, EEYP mutant channels might remain in a relatively stable state in the ER, Golgi, or other compartment along the trafficking pathway and may not undergo rapid degradation.
To limit the mechanisms that might apply, degradation products were evaluated, in HCN2-4A (the full-length representative of the EEYP mutant phenotype) as compared with HCN2 and a set of HCN2 NH2-terminal deletion mutants characterized by altered trafficking, enhanced shunting of channel protein to the degradation pathway and diminished production of current (unpublished observations). HCN2-2-137 (NH2-terminal amino acids 2-137 removed) was first used in this analysis because it produces levels of If comparable to HCN2-4A. Representative current traces for the NH2-terminal mutant HCN2-2-137 and HCN2-4A mutant are shown in comparison to wild-type HCN2, in Fig. 6A. In both mutants a significant and comparable reduction in If density relative to the wild-type channel was confirmed (Fig. 6B); If activation is unaltered (Fig. 6C).
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2-137, and HCN2-N380Q channels by Western blotting, using a COOH-terminally directed antibody for HCN2 (Fig. 6D). Mature and immature forms were again identified at 136 kDa and 114 kDa for HCN2, whereas only the immature form was identified for HCN2-4A and HCN2-N380Q. In HCN2-2-137, a band corresponding to an immature form (83 kDa), and an obvious smaller band at 53 kDa were present (Fig. 6D). A band at this lower molecular mass was also found in lanes containing HCN2, HCN2-N380Q, and HCN2-4A, but at very low intensity. This 53-kDa band is likely a degraded form of HCN2, and its greater intensity in the lane containing HCN2-2-137 suggests that this mutant undergoes degradation to a greater extent. Successively larger truncations of the NH2 terminus progressively engage the degradation pathway and decrease If density (Fig. 7). This demonstrates that the level of cell surface expression of the NH2-terminal truncation mutants parallels the extent of observed degradation. Thus, the efficiency of channel folding and assembly of these NH2-terminal deletion mutants is likely decreased. On the contrary, for HCN2-4A, there is a low level of degradation observed that is comparable to the wild-type channel. Thus, the roughly equivalent reduction in functional expression of the HCN2-4A and NH2-terminal truncations must therefore come about by different mechanisms. The EEYP mutant channels, which are ostensibly immature, likely reside in an intracellular compartment in relatively stable state. The similar lack of degradation seen in the HCN2-N380Q mutant, which too is immature, suggests that a trafficking step that involves complex glycosylation may be regulated by the EEYP motif. As for the HCN2-4A, bands corresponding to the mature form of the two NH2-terminal mutant channels were apparent when four times more protein was loaded (data not shown); this corresponds to the lower levels of current measured and suggests that the mutant channels were retained to a greater extent compared with wild-type channels.
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40%, double alanine mutation of the second two amino acids (YP) reduces current by >60%, and replacement of all four amino acids by alanine reduces current by almost 90%. These data suggest that "YP" contributes to current levels to a greater extent than "EE" and that the combined effects of both couplets are almost completely additive.
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DISCUSSION |
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A separate region of the CNBD was delimited that is responsible for inhibition of channel opening, namely, the A-helix, β-barrel, and proximal B-helix. It will be important to determine the precise element required for this function. Inhibition involves interactions of the COOH terminus with the transmembrane domains (30, 31), but the exact molecular determinants and nature of the interactions are not known. Although the EEYP does not contribute to channel inhibition, small negative shifts in If activation were observed in cells expressing HCN2-4A, HCN2-AAYP and HCN2-EEAA, but not in cells expressing the truncated HCN2-EEYP-C-term or HCN2-4A-C-term. These data suggest that the EEYP may influence opening of the channel by interacting with downstream elements of the COOH terminus. The C-helix, distal and adjacent to the B-helix, is a probable candidate for this interaction since it is required to transduce the actions of cAMP on channel opening (31).
Important clues about the mechanism underlying enhancement of cell surface expression by the EEYP motif are presented. In this study, the observation that functional HCN2 channels can form in the absence of EEYP, albeit at low levels, shows that they are not irreversibly misfolded. Thus, this motif is not an absolute requirement for correct folding, assembly, and delivery of functional channels to the cell surface. Instead, the EEYP motif likely regulates the efficiency of cell surface expression. Unlike HCN2 NH2-terminal truncation mutants, the levels of degradation of HCN2-4A, as determined using antibodies directed to the COOH terminus, were comparable to wild-type, which points toward one of two possibilities. First, the channels may be identified as misfolded and shunted toward a pathway in which degradation does not occur rapidly. To identify misfolded proteins, cells possess multiple folding "checkpoints" and pathways (2, 8, 9, 13), but only some of these are associated with their rapid clearance and degradation (10, 29). Thus, EEYP mutant channels may be misfolded but in a relatively stable state intracellularly. Second, correctly folded and assembled EEYP mutant channels may reside in an intracellular compartment in a relatively stable state (15, 17, 24) and thus may likewise not undergo rapid degradation. The EEYP may actively promote the movement of these correctly folded and assembled channels out of the ER or actively reduce channel retention in the ER, possibly by masking a retention signal. The lack of degradation of the EEYP mutants may be related to the observation that the EEYP motif does not impact the spectrum of oligomeric assembly of intracellular channels, but a determination of whether the intracellularly localized channels are folded and assembled correctly is required to discriminate between the two proposed possibilities.
Because the EEYP mutants reduced but did not abolish functional expression, this motif is not the sole determinant of HCN2 cell surface expression. A proximal sequence in the CNBD and/or extant regions is likely of added importance. Interestingly, the primary structure of the entire B-helix region (VDNFNEVLEEYP) contains a number of acidic amino acids—an important feature of anterograde signals that facilitate forward trafficking but do not affect folding or assembly (6, 14, 15, 17, 24). It is also interesting that HCN4 but not HCN2 channels devoid of the CNBD are able to form functional channels in another mammalian cell line (HEK) (25). This is further evidence that regions other than the EEYP, which is conserved among all of the mammalian isoforms, and the CNBD are involved in the regulation of HCN cell surface expression.
Unlike the EEYP mutants and wild type channels, the NH2-terminal truncation mutants were subject to significant degradation when expressed in CHO cells, despite the fact that the levels of If for some of the truncation mutants were similar to those of the EEYP mutants. This suggests that the similar reductions in If produced by NH2-terminal truncations and EEYP mutants were the result of different processes. Previously, we proposed that the NH2 terminus plays a role in channel assembly similar to that of the NH2-terminal T1 domains in Shaker-related voltage-gated potassium channels (20, 26). In Shaker channels, T1 domains facilitate tetramerization by bringing subunits into close proximity at the start of their assembly, which begins while still attached to ribosomes (12), and thereby increase the effective local concentration of compatible subunits (33). This is consistent with findings that show that T1-deleted Shaker-related channels require high mRNA concentrations and prolonged times to form channels (11, 27). Taken together, the data suggest that HCN2 subunits lacking portions of the NH2 terminus are identified by a distinct mechanism and degraded during an inefficient folding and assembly process.
A revealing finding in our study is that different defects within the same channel, which produce similar reductions in cell surface expression, lead to different end points in a given cell. This is consistent with previous studies that have demonstrated that the location of the lesion within a protein determines the predominant pathway used for protein disposal and that the pathway chosen depends on the ability of the protein to pass through several sequential checkpoints (29). This has important implications for disease-associated mutations in HCN channels and other channels of the voltage-gated potassium channel superfamily. A mutation in the COOH terminus has been proposed to disrupt cell surface expression of HCN4 and cause sinoatrial arrhythmia (28). In addition, many disease-associated mutations in related channels such as the human ether-a-go-go, KCNQ1, and cyclic nucleotide-gated channels lead to disruption of cell surface expression (18, 19, 23). Our findings suggest that cell surface disruption may occur by mutation and defect-specific mechanisms, which then lead to consequences unique to those defects. Multiple mechanisms of protein disposal, and their diverse consequences, likely contribute to the variable and pleiotropic nature of disease-associated mutations with this superfamily of channels. An important future goal will be to elucidate the molecular and cellular details of these diverse mechanisms, and to relate these to specific mutations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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and k, values were not significantly different among the channels (one-way ANOVA, P > 0.05). D: Western blot probed with a rabbit antibody specific for the mammalian HCN2 COOH terminus. lane 1, untransfected cells; lane 2, HCN2; lane 3, HCN2-N380Q; lane 4, HCN2-4A; lane 5, HCN2-
