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Cardiovascular Research Advance Access first published online on March 7, 2008
This version [Corrected Proof] published online on March 31, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn062
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Recycling endosomes supply cardiac pacemaker channels for regulated surface expression

Nadine Hardel, Nadine Harmel, Gerd Zolles, Bernd Fakler and Nikolaj Klöcker*

Institute of Physiology, University of Freiburg, Hermann-Herder-Str. 7, 79104 Freiburg, Germany

* Corresponding author. Tel: +49 761 203 5141; fax: +49 761 203 5191. E-mail address: nikolaj.kloecker{at}physiologie.uni-freiburg.de

Received 23 October 2007; revised 18 February 2008; accepted 2 March 2008

Time for primary review: 22 days


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
Aims: Cellular excitability is not only determined by the type but also by the number of ion channels in the plasma membrane. Recent evidence indicates that cell surface expression of cardiac pacemaker channels might be controlled beyond the level of biosynthesis by regulating their surface transport. However, neither the underlying trafficking pathways nor their molecular control have yet been investigated.

Methods and results: We have studied endocytic trafficking of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels expressed as fusions with green fluorescent protein or tagged with an extracellular haemagglutinin epitope in opossum kidney cells, dissociated rat hippocampal neurons, and ventricular cardiomyocytes. After being internalized from the plasma membrane, HCN2 and HCN4 are sorted to the Rab11-positive endocytic recycling compartment (ERC). From there, they are transported back to the cell surface depending on active phospholipase D2 (PLD2). The peptide hormone angiotensin II, which is upregulated in a number of cardiac pathologies and a known activator of PLD2, stimulates ERC trafficking of HCN4 channels. It significantly increases HCN surface expression independent of their biosynthesis.

Conclusion: Recycling endosomes serve as an intracellular storage compartment for the cardiac pacemaker channels HCN2 and HCN4. They are not only crucial for maintaining a homeostatic surface expression but also supply channels for rapid adaptation of their surface expression in response to extracellular stimuli.

KEYWORDS HCN channel; Endocytic recycling compartment; Phospholipase D2; Angiotensin; Insulin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
The hyperpolarization-activated cation current If contributes significantly to pacemaker activity in the mammalian heart.1,2 Stimulation of the sympathetic nervous system accelerates heart rate, among others, by activating If via a cAMP-dependent shift in its activation curve to more depolarized potentials.3 Increases in If can lead to enhanced automaticity and have thus been implicated in the aetiology of cardiac arrhythmias.4 If is conducted by members of the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel family. Four mammalian HCN genes (HCN1-4) have been identified which show distinct expression patterns in the heart and brain.5,6 Although they also have distinct biophysical properties with respect to channel kinetics and cyclic nucleotide gating,7 they share the characteristic that their reliable activation in the subthreshold voltage range of cardiomyocytes and neurons requires their interaction with membrane phospholipids.8,9

Cellular excitability is not only determined by the type but also by the number of ion channels in the plasma membrane. Surface expression of ion channels and other integral membrane proteins may be controlled beyond the level of protein biosynthesis by regulating their transport along the secretory pathway.10,11 Thus, a number of intrinsic sequence motifs have been characterized that govern export of channel proteins from the endoplasmic reticulum (ER) or the Golgi complex. In addition, surface expression of ion channels can be regulated by dynamic control of their endocytosis and exocytosis from intracellular storage compartments. One such storage compartment is the endocytic recycling compartment (ERC), a peri-nuclear collection of tubular organelles that mature from sorting endosomes.12 Protein traffic through the ERC is poorly understood; two proteins that specifically regulate cargo export from the ERC are the GTPase Rab11 and the Eps15-homology-domain protein EHD-1/Rme-1, which also serve as marker proteins for this compartment.12,13 Moreover, the ubiquitously expressed phospholipase D (PLD), whose activity is elevated in response to activation of a number of hormone, neurotransmitter, and growth factor receptors,14,15 has been tightly linked to the regulation of endocytic traffic through the ERC.16

Recent studies reported on modulations of the amplitude of Ih, the brain analogue of If, that are mediated by changes in HCN channel surface expression.17–19 Thus, activity-dependent changes in Ih current density have been observed on time scales faster than expected for mere increases in protein biosynthesis indicating changes in channel surface transport. Here, we have investigated the subcellular trafficking of HCN channels in more detail. Our results demonstrate that recycling endosomes maintain surface expression of the cardiac pacemaker channels HCN2 and HCN4 and allow its dynamic adaptation upon extracellular signalling.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
2.1 Molecular biology
N-terminal fusion constructs of HCN channels with enhanced green fluorescent protein (EGFP) were designed by inserting the respective cDNA (mHCN1, NM_010408 [GenBank] ; mHCN2, NM_008226 [GenBank] ; mHCN3, BC039156 [GenBank] ; hHCN4, NM_005477 [GenBank] ) in-frame into EGFP-C1-3 expression plasmids (BD Biosciences). Mutagenesis was performed as described elsewhere20 and verified by sequencing. The haemagglutinin (HA) epitope plus flanking residues were introduced into the extracellular domains of mHCN2 and hHCN4 at amino acid positions 284 and 362, respectively, reading 284G-ISAYGITYPYDVPDYA-I285 in HCN2 and 362R-ISAYGITYPYDVPDYA-I363 in HCN4.21 Rab11 and Rme-1 constructs were expressed as GFP fusions,13 PLD2 constructs were in pcDNA3.1.22 The Golgi complex was identified by expressing a fusion construct of the 81 N-terminal amino acids of the Golgi-resident enzyme β-1,4-galactosyltransferase and GFP.23

2.2 Cell culture and transfection
Opossum kidney (OK) cells and COS-7 (American Type Culture Collection) cells were used as heterologous expression systems with a large cytosol-to-nucleus ratio facilitating the identification of subcellular compartments. OK cells were grown in DMEM-F12 supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin (P/S; Invitrogen) at 37°C and 5% CO2. COS-7 cells were grown at the same conditions in DMEM supplemented with 10% FCS, 1% HEPES, and 1% P/S. At ~80% confluence, cells were transfected with the respective cDNAs using Lipofectamine 2000 (Invitrogen) following the supplier’s directions. The following drugs were applied for times indicated in the results section: brefeldin A (BFA; 10 µg/mL), cycloheximide (CHX; 50 µg/mL), phorbol-12-myristate-13-acetate (PMA; 100 nM), angiotensin II (ATII; 100 nM), insulin (100 nM) (all purchased from Sigma), and the protein kinase C (PKC) inhibitor Ro-31-8425 (Ro; 100 nM; Calbiochem). For blocking the ATII receptor subtypes AT1R and AT2R, ZD7155 (100 nM; Tocris Bioscience) and PD 123319 (2 µM; Sigma) were used, respectively.

Primary cultures of ventricular cardiomyocytes were prepared from neonatal rats. Briefly, ventricular cells were dispersed in 0.3% trypsin in HBSS (Invitrogen). Cells were pre-plated for 90 min to enrich cultures for myocytes (90–95% of the cells). Cardiomyocytes were grown on laminin-coated coverslips in DMEM supplemented with 10% FCS (0.2% after DIV2) and 1% P/S. For electroporation of cardiomyocytes, approximately 400 000 cells were incubated with 4 µg of the respective cDNA in 300 µL medium and electroporated at 420 V, 75 µF in an Easyject Optima electroporator (Equibio). Cardiomyocytes were identified by the striated aspect of their actin cytoskeleton stained with TRITC-conjugated phalloidin (Sigma).

Primary cultures of hippocampal neurons were obtained from rats at P0. The entire hippocampus was isolated and dissociated with papain, and cells were plated in a 24-well plate at a density of approximately 80 000 cells/well in neurobasal medium (Invitrogen) supplemented with B27, L-glutamine, and 1% P/S. Hippocampal neurons were grown at 37°C in 5% CO2 on coverslips coated with poly-D-lysine and collagen (Invitrogen). They were transfected on DIV7 with Lipofectamine 2000 according to the manufacturer’s recommendations except that 1.2 µg cDNA in 25 µL Opti-MEM and 0.7 µL of Lipofectamine 2000 in 25 µL Opti-MEM were mixed and added to coverslips. Two days after transfection, cells were processed for immunocytochemistry.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Immunocytochemistry and transferrin uptake
For immunocytochemistry, transfected cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at 4°C and pre-treated with 10% normal goat serum in PBS with 0.04% Triton X-100 (PBS-T) for 1 h at room temperature (RT) to block unspecific antibody binding. Then they were incubated with the respective primary antibody diluted in 2% NGS/PBS-T for 1 h at RT. Immunoreactivity was finally visualized by goat anti-mouse antiserum conjugated to cy-2 or cy-3 (1:250 in 10% NGS/PBS-T). To detect selectively the population of HA-tagged channels expressed on the cell surface or for internalization assays, antibody staining was performed in vivo without use of detergents. Unless otherwise stated, antibody incubation was carried out in serum-free medium for 30 min at 37°C for the primary and secondary antiserum, respectively. This in vivo labelling approach often led to a clustered surface distribution of the respective constructs which is artificially induced by antibody cross-linking.23 The following primary antibodies were used: monoclonal mouse anti-HA (Santa Cruz Biotechnology; 1:100), monoclonal mouse anti-MAP2 (Sigma; 1:100).

Transferrin uptake assays were performed by incubation of COS-7 cells with fluorescently labelled transferrin (Invitrogen; 30 µg/mL serum-free medium) for 30 min at 37°C.

2.4 Quantification of surface expression
Surface expression of extracellularly tagged HCN channels was quantified by fluorescence intensity measurements of anti-HA immunocytochemistry without use of detergents as described before.23 For each construct, pixel intensity values of stainings from at least two or more independent transfections were corrected for background and integrated over 20 areas of 0.2116 mm2 each (ImageJ, NIH). Test and control groups were always processed in parallel to correct for differences in staining efficiency between experiments. Only samples with fluorescence intensities that were well in the dynamic range of the assay were used for data analysis using Igor Pro (Wavemetrics). Statistically significant differences (asterisks indicate P < 0.01) were assessed using the unpaired Student’s t-test. Data are given as mean ± SEM expressed as relative surface expression level of the respective control (for dominant-negative proteins, the control was the respective wild-type protein). Such approach was chosen to exclude potential effects of co-expressed constructs on the protein expression level of HCN channels.

2.5 Imaging
Cells were imaged with a confocal laser scanning microscope (LSM510, Zeiss) using the following excitation wavelengths and filter settings. EGFP, cy-2, transferrin-488: excitation 488 nm Ar laser/emission BP505-530 nm; cy-3, transferrin-543: excitation 543 nm He laser/emission LP560 nm.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
3.1 Subcellular distribution of hyperpolarization-activated cyclic nucleotide-gated channels
When heterologously expressed in OK cells, the four HCN channel subtypes exhibited distinct patterns of subcellular distribution (Figure 1). GFP-fused HCN4 (GFPHCN4) appeared concentrated in a juxta-nuclear compartment, whereas GFPHCN3 was homogenously distributed throughout the ER (see Supplementary material online, Figure S1). GFPHCN1 and GFPHCN2 showed intermediate patterns of distribution, with only little accumulation in the peri-nuclear region (HCN2 > HCN1). On the basis of the assumption that primary sequence information caused the subcellular segregation of HCN3 and HCN4, the HCN4 C-terminus was deleted downstream of the cyclic-nucleotide binding domain (CNBD). Where such deletion converted the subcellular localization pattern of GFPHCN4 into the one of GFPHCN3 (HCN4-{Delta}CT; Figure 1), we found that fusion of it to GFPHCN3 at homologous position let the chimeric channel (HCN3/4CT) concentrate in the juxta-nuclear compartment. This indicated that sequence information contained within the HCN4 C-terminus is not only necessary but also sufficient for peri-nuclear accumulation of the channel protein.


Figure 1
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  		Figure 1 Subcellular distribution of hyperpolarization-activated cyclic nucleotide channel isoforms in epithelial cells. (A) Representative confocal images of opossum kidney cells expressing the indicated green fluorescent protein-fused hyperpolarization-activated cyclic nucleotide channel isoforms and mutants. Note the peri-nuclear accumulation of HCN4 which is lost in its C-terminal deletion mutant ({Delta}CT, {Delta}716-1203) and transferred to HCN3 in the chimera HCN3/4CT. Scale bar: 10 µm. (B) Schematic drawing of the C-terminal deletion and chimeric constructs. Numbers/letters indicate amino acids in the primary sequences. Core, ion channel core domain with six transmembrane (TM) helices; CNBD, cyclic nucleotide-binding domain.

 
The observed differences in subcellular localization of the respective HCN subtypes were neither due to N-terminal GFP fusion, as extracellularly HA-tagged HCN channels showed similar distributions (see Supplementary material online, Figure S2), nor were they caused by simple overexpression, as incubation of cells with the proteasome inhibitor acetyl-L-leucyl-L-leucyl-norleucinal did not affect the subcellular channel distribution (data not shown). In addition, both N-terminal GFP fusion and extracellular HA-tagging did prevent neither formation nor surface traffic of functional HCN channel complexes as demonstrated by electrophysiological means (see Supplementary material online, Figure S3).

Finally, we could exclude a bias of the heterologous expression system, as HCN3 and HCN4 showed their distinct subcellular localizations also in a primary cell background. Both in neonatal ventricular cardiomyocytes and in hippocampal neurons, GFPHCN4 accumulated in the peri-nuclear region (in neurons also along dendrites), whereas GFPHCN3 was homogenously distributed throughout the cells (Figure 2).


Figure 2
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  		Figure 2 Peri-nuclear accumulation of HCN4 is preserved in primary cells. Representative confocal images of dissociated neonatal ventricular cardiomyocytes (A) and hippocampal neurons (B and C) expressing green fluorescent protein-fused HCN3 and HCN4. Cardiomyocytes were identified by their striated actin cytoskeleton visualized by fluorescent phalloidin; neurons were identified by anti-MAP2 immunocytochemistry. Note that HCN4 shows peri-nuclear (A and B) and dendritic (C) accumulations, whereas HCN3 is homogenously distributed.

 
By employing an extracellular epitope-tagging approach, we characterized in more detail the juxta-nuclear compartment in which HCN4 channels accumulated. Thus, channel protein expressed on the cell surface was immunolabelled in vivo with a primary antibody directed against the artificially inserted extracellular HA-epitope. After channels had been allowed to internalize, two fluorescent secondary antisera were applied before and after membrane permeabilization to distinguish between channels still present on the cell surface and channels which had entered endocytic compartments. As shown in Figure 3, HCN4 was constitutively internalized from the plasma membrane into the respective peri-nuclear compartment, indicating its role as an endosomal compartment involved in retrograde protein traffic or recycling. Within this compartment, internalized HCN4 (HCN4i) co-localized with internalized transferrin, with the GTPase Rab11, and with the Eps15-homology-domain protein EHD-1/Rme-1, strongly suggesting accumulation of HCN4 in the so-called endocytic recycling compartment (ERC). In good agreement with this hypothesis, we found the distribution of internalized HCN4 to be insensitive to the fungal toxin BFA in contrast to the partially co-localizing Golgi marker protein β-1,4-galactosyltransferase23 which redistributed into the ER upon BFA treatment.


Figure 3
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  		Figure 3 HCN4 is internalized into the endocytic recycling compartment. Representative confocal images of opossum kidney cells expressing extracellularly haemagglutinin-tagged HCN4 (scheme of the haemagglutinin-tagged channel protein on top). The extracellular epitope-tagging approach allowed to distinguish between channels expressed on the cell surface (HCN4s) and internalized channels (HCN4i). For further details, see Materials and methods. Retrograde transport of HCN4 into the endocytic recycling compartment was identified by peri-nuclear co-localization of internalized HCN4 (HCN4i) with a number of marker proteins for this compartment: internalized transferrin-Alexa488, GFPRab11, and GFPRme-1. In contrast to the Golgi marker,22 accumulation of HCN4i was resistant to brefeldin A treatment (10 µg/mL; 45 min at 37°C). Scale bar: 10 µm; 6 µm for the co-localization experiment with the Golgi marker without brefeldin A.

 
3.2 Recycling through the endocytic recycling compartment maintains hyperpolarization-activated cyclic nucleotide-gated channel surface expression
To probe the functional significance of the ERC for HCN channel trafficking, we co-expressed the channels with wild-type and dominant-negative mutants of proteins interfering with ERC function and measured surface expression of channel proteins by means of extracellular epitope tagging. Co-expression of wild-type Rab11, Rme-1, and PLD2 did not significantly change steady-state surface expression of HCN4 co-expressed with GFP as control (data not shown). However, the dominant-negative mutants Rab11-S25N, Rme-1-G429R, and PLD2-K758R significantly reduced steady-state surface expression of HCN4 channels to 41 ± 4%, 68 ± 3%, and 65 ± 5% of control cells co-expressing the respective wild-type protein (Figure 4A). Likewise, HCN2 channel surface expression was reduced to 51 ± 4%, 59 ± 2%, and 57 ± 2% upon co-expression of mutant Rab11, Rme-1, and PLD2, respectively (Figure 4B). Total HCN protein expression remained unaffected by co-expression of the respective dominant-negative mutants (see Supplementary material online, Figure S4).


Figure 4
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  		Figure 4 Recycling endosomes supply hyperpolarization-activated cyclic nucleotide channels for maintaining their surface expression. Surface expression of HCN4 (A) and HCN2 (B) is significantly reduced upon co-expression of dominant-negative proteins as indicated. Left: representative images of opossum kidney cells in which hyperpolarization-activated cyclic nucleotide channels expressed on the cell surface were detected by extracellular epitope tagging; fluorescence intensity is colour-coded: red represents high expression, blue represents low expression. Right: quantification of hyperpolarization-activated cyclic nucleotide surface expression. (C) Sequence information contained in the HCN4 C-terminus directs the channel into the endocytic recycling compartment. Left: surface expression of HCN4-{Delta}CT ({Delta}716-1203) upon co-expression of indicated proteins. Right: surface expression of wild-type HCN4 and indicated C-terminal deletions upon co-expression of dominant-negative Rab11S25N. Quantitative data in (A)–(C) are mean surface expression levels ± SEM given in percentage of control (co-expression of the respective wild-type protein). Asterisk marks a significant difference from control (P < 0.01; n = 20).

 
In contrast to HCN4 wild-type, steady-state surface expression of the C-terminal deletion construct of HCN4 (HCN4-{Delta}CT), which did not accumulate within the ERC but reached efficiently the plasma membrane, remained unaffected by co-expression of dominant-negative Rab11 (95 ± 5%), Rme-1 (110 ± 7%), or PLD2 (93 ± 5%) (Figure 4C, left). In an attempt to narrow down the C-terminal sequence stretch both necessary and sufficient for HCN4 cycling through the ERC, we tested increasing C-terminal deletions of HCN4 for the loss of Rab11-S25N-induced reduction in steady-state surface expression. As illustrated in Figure 4C (right), such loss was observed in HCN4-{Delta}816-1203 (99 ± 7%) but not in HCN4-{Delta}907-1203 (46 ± 3%), positioning the crucial sequence stretch for ERC cycling between amino acids 816 and 907. Further delineation of the sequence by deletion analysis and transfer experiments onto HCN3 was not successful, indicating that it might not be a simple linear signal.

3.3 Activation of PLD2 by extracellular stimuli increases HCN4 channel surface expression
As inhibition of PLD2 activity by co-expressing a dominant-negative mutant had decreased surface expression of HCN2 and HCN4, we next tested whether stimulation of PLD2 activity would enhance channel recycling from the ERC to the plasma membrane.

PLD can be activated by PMA via activation of PKC.16,24 Incubation of cells with 100 nM PMA for 1 h significantly increased surface expression of HCN4 by 62 ± 4%, an effect that was independent of protein biosynthesis, as pre-incubation with CHX (50 µg/mL) for 1 h did not attenuate the effects of PMA (150 ± 7% of control) (Figure 5A). In contrast, both the PKC inhibitor Ro-31-8425 (Ro; 100 nM, 1 h) and co-expression of the dominant-negative mutant of PLD2 prevented the PMA-induced increase in HCN4 surface expression (110 ± 6 and 109 ± 6%, respectively).


Figure 5
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  		Figure 5 Surface expression of HCN4 is increased upon stimulation of endocytic recycling compartment transport. Quantification of surface expression levels of HCN4 upon treatment of cells with 100 nM PMA (A), 100 nM ATII (B), and 100 nM insulin (C and D) for 1 h. Other drugs were applied as indicated. Note that the increase in HCN4 protein on the cell surface induced by PMA and ATII was not prevented by CHX in contrast to the effects of insulin on HCN4 surface expression. Upon co-expression of dominant-negative PLD2 (K758R), neither PMA nor ATII was able to increase surface expression of HCN4. Data in (A)–(C) are mean surface expression levels ± SEM given in percentage of control (white bars). (D) Co-expression of the respective wild-type protein served as control. '*' marks a significant difference from control (P < 0.01; n = 20), '#' marks a significant difference from PMA or ATII-treated groups (P < 0.01; n = 20). PMA, phorbol-12-myristate-13-acetate 100 nM; CHX, cycloheximide 50 µg/mL; Ro, Ro-31-8425 100 nM; ATII, angiotensin II 100 nM.

 
The hormone ATII is centrally involved in a number of cardiac pathologies including chronic heart failure (CHF) and diabetic cardiomyopathy. ATII signals through two types of receptors, of which AT1R is known to stimulate PKC and PLD by coupling to Gq-protein,25,26 whereas AT2R signalling is far less understood.27,28 As shown in Figure 5B, incubation of cells with 100 nM ATII for 1 h resulted in a significant increase in HCN4 steady-state surface expression by 64 ± 5%, which was unaffected by incubation with CHX (171 ± 4% of control). As was the case for PMA, both the PKC inhibitor Ro-31-8425 (Ro) and co-expression of the dominant-negative mutant of PLD2 abolished the effect of ATII (126 ± 8 and 95 ± 2%, respectively). The ATII effect on HCN4 surface expression was also significantly reduced by both the AT1R blocker ZD7155 (100 nM; 141 ± 3%) and the AT2R blocker PD123319 (2 µM; 110 ± 5%).

Finally, we tested whether insulin, another hormone relevant for cardiac disease and also reported to stimulate PLD2 activity,29 would change steady-state HCN4 surface expression. As depicted in Figure 5C, we observed a significant increase of surface HCN4 by 78 ± 9%. However, the insulin-mediated increase in HCN4 surface expression was sensitive to CHX treatment, indicating that it was rather due to increased synthesis and not to increased recycling of channel proteins. Sensitivity of insulin-mediated increase in HCN4 surface expression to inhibition of ERC transport by co-expression of Rab11-S25 N, Rme-1-G429R, and PLD2-K758R was preserved (Figure 5D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Supplementary material
 Funding
 References
 
The central issue of the present work is characterizing the endocytic trafficking behaviour of the cardiac pacemaker channel subunits HCN2 and HCN4. After having reached the plasma membrane, channel proteins are internalized and sorted into recycling endosomes which serve as a storage compartment for both maintaining and dynamically adapting channel surface expression in response to extracellular stimuli.

Upon expression both in a heterologous and a native cell background, HCN4 and to lesser extent also HCN2 and HCN1 channels accumulated in a peri-nuclear compartment. We identified this compartment as the ERC, as (i) the channel was sorted into the compartment after having been internalized from the plasma membrane, (ii) internalized channel protein co-localized with the ERC markers Rab11 and Rme-1,12 and (iii) the subcellular distribution of the internalized protein was resistant to BFA treatment in contrast to the partially co-localizing Golgi marker which dispersed into the ER.30,31 The ERC is a peri-nuclear collection of tubular organelles that serve important sorting functions in recycling plasma membrane and its protein constituents.12 Once having reached the sorting endosomes, internalized membrane proteins use basically two main routes for returning to the cell surface: they are either transported directly back to the plasma membrane or follow a slower pathway through the ERC. Neither the molecular determinants that distinguish between these two pathways nor the ones that control export from the ERC have been well defined yet. Mutagenesis studies on the transferrin receptor (TfR), the prototype protein for investigating endocytic recycling through the ERC, challenge a general significance of protein determinants in ERC passage. Thus, the deletion of the cytoplasmic domain of the TfR does not change its intracellular recycling itinerary after internalization.32 However, other transmembrane receptors such as the β2-adrenergic and the µ-opioid receptors do depend on cytoplasmic signals such as (acidic cluster) di-leucine motifs for directing their recycling traffic properly.33,34 Here, we show that HCN4 also contains sequence information downstream of the CNBD (between residues 816 and 907) that is both necessary and sufficient for efficient import of the channel protein into the ERC. A more detailed analysis of the isolated proline-rich sequence stretch with respect to protein interaction partners will help to elucidate its molecular mechanism of action.

The ERC pathway plays a major role in maintaining the homeostasis of HCN channel surface expression, as the latter was reduced by ~50% upon co-expression of dominant-negative Rab11 and Rme-1 which block ERC exit. These findings agree well with recent studies showing that surface numbers of other ion channels also depend largely on Rab11-dependent endosomes.35–38 In addition to maintaining the steady-state protein composition of the plasma membrane, ERC transport rates might be subject to change in response to signalling mechanisms in order to dynamically adapt surface expression of membrane proteins.36 The ubiquitously expressed phospholipases of the PLD family, whose basal activity can be stimulated via PKC activation by various hormone and growth factor receptors, have been implicated in controlling endosomal traffic.14,15 PLD enzymes hydrolyse phosphatidylcholine to yield free choline and phosphatidic acid (PA). PA can then act as a lipid anchor for translocating endosomal trafficking proteins to the plasma membrane,39 it can activate enzymes of the phosphatidylinositolkinase family which are involved in vesicle trafficking,40,41 and it was ascribed fusogenic properties.42,43 In line with the view that the PLD2 isoform is centrally involved in ERC export,16 we observed a significant decrease in surface expression of HCN2 and HCN4 channels upon co-expression of dominant-negative PLD2, whereas surface expression of the C-terminal deletion mutant of HCN4, which does not enter the ERC pathway, remained unaffected. We could further demonstrate that stimulation of PLD activity by PMA-mediated PKC activation significantly increased surface expression of HCN4, an effect that was independent of protein biosynthesis but was readily blocked by the dominant-negative PLD2 mutant. These results suggest that HCN channel surface expression is under regulatory control of PLD2 activity.

A number of cardiac pathologies including CHF and diabetic cardiomyopathy involve activation of the renin–angiotensin system (RAS) which eventually increases the production of ATII. This hormone acts on two types of receptors, with the AT1R activating PLD via PKC stimulation.25,26 Here, we found that ATII significantly increased surface expression of HCN4 channels in a PLD2-dependent manner. Intriguingly, both the AT1R inhibitor ZD7155 and the AT2R blocker PD123319 were effective in reducing the ATII-mediated increase in HCN4 surface expression. The AT2R signal transduction pathways are still a matter of debate but seem to involve the activation of various protein phosphatases, the activation of the NO/cGMP system, and stimulation of phospholipase A2 (PLA2).27,28 Experimental evidence indicates that PLA2 might activate PLD2 through generation of lipid intermediates.44 Thus, signalling of both AT1R and AT2R might eventually converge on PLD2 activation which would explain the complete inhibition of the ATII-induced increase in HCN4 surface expression by dominant-negative PLD2. In CHF, increases in If current densities in cardiomyocytes have been described45–47; such increase might significantly contribute to cardiac arrhythmias frequently associated with CHF in patients. Our study provides a molecular hypothesis as to how RAS activation due to left ventricular dysfunction might result in an increase in If current density, that is, by enhancing export of HCN channels from the ERC. Increased recycling of HCN channels might also provide a molecular explanation for the acute chronotropic effects of ATII that are independent of arterial blood pressure levels.48,49

Prompted by both clinical and experimental studies showing that insulin-dependent diabetes is associated with an impaired regulation of heart rate, we investigated whether the hormone insulin, also reported to activate PLD2,29 would increase cell surface traffic of HCN channels. Indeed, we found that insulin induced a significant increase in HCN4 surface expression. Unlike for ATII, however, this increase was completely abolished by CHX, indicating that the insulin effect on HCN4 channel surface expression relied most likely on the biosynthesis of new channel protein. It did not involve enhanced trafficking of pre-formed channels from intracellular stores as described for the glucose transporter GLUT4 or N-methyl-D-aspartate receptors.50,51


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Supplementary material
 Funding
 References
 
In summary, we have shown that Rab11-positive recycling endosomes supply HCN channels for both maintaining their steady-state surface expression and adapting it in response to extracellular stimuli. As the involved PKC and PLD2-dependent pathways are well conserved across cell types, this work provides a general molecular mechanism as to how the density of the hyperpolarization-activated cation current If (Ih) might be controlled by regulated surface transport of HCN channels.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
References
 
Deutsche Forschungsgemeinschaft (KL-1168/6 to N.K. and GRK-843).


   Acknowledgements
 
The authors would like to thank I. Bierschenk and G. Kummer for excellent technical assistance. HCN channel cDNAs were kindly provided by B. Santoro (Columbia University) and A. Ludwig (University of Erlangen); the PLD2 constructs were a gift from T. Koch (University of Magdeburg); Rme1 and Rab11 including their dominant-negative mutants were kindly provided by B. Grant (Rutgers University).

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 

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