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• W2023243905 abstract "Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity. Although identified only recently, the family of hyperpolarization-activated cyclic nucleotide-gated (HCN) 3The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-gated channels; PTK, protein-tyrosine kinase; SH, Src homology; YTH, yeast two-hybrid; GST, glutathione S-transferase; HEK, human embryonic kidney; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; PBS, phosphate-buffered saline; PI, protease inhibitor mix; MOPS, 4-morpholinepropanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; DRG, dorsal root ganglion; CNBD, cyclic nucleotide binding domain. channels has generated great interest because it represents the molecular correlate of the hyperpolarization-activated cation current, termed Ih (syn. If or Iq) (1Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (985) Google Scholar, 2Kaupp U.B. Seifert R. Annu. Rev. Physiol. 2001; 63: 235-257Crossref PubMed Scopus (307) Google Scholar, 3Biel M. Schneider A. Wahl C. Trends. Cardiovasc. Med. 2002; 12: 206-212Crossref PubMed Scopus (210) Google Scholar, 4Robinson R.B. Siegelbaum S.A. Annu. Rev. Physiol. 2003; 65: 453-480Crossref PubMed Scopus (916) Google Scholar). This current plays a crucial role in the control of important biological functions, including cardiac and neuronal pacemaker activity, determination of resting membrane potential, dendritic integration, and synaptic transmission. Dysfunction of HCN channels has been linked to human diseases, including cardiac arrhythmia (5Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (314) Google Scholar, 6Ueda K. Nakamura K. Hayashi T. Inagaki N. Takahashi M. Arimura T. Morita H. Higashiuesato Y. Hirano Y. Yasunami M. Takishita S. Yamashina A. Ohe T. Sunamori M. Hiraoka M. Kimura A. J. Biol. Chem. 2004; 279: 27194-27198Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and epilepsy (7Bender R.A. Soleymani S.V. Brewster A.L. Nguyen S.T. Beck H. Mathern G.W. Baram T.Z. J. Neurosci. 2003; 23: 6826-6836Crossref PubMed Google Scholar, 8Poolos N.P. Epilepsy. Curr. 2004; 4: 3-6Crossref PubMed Google Scholar). Structurally, the four members of the HCN channel family (HCN1-4) belong to the 6TM ion channel superfamily (9Gauss R. Seifert R. Kaupp U.B. Nature. 1998; 393: 583-587Crossref PubMed Scopus (380) Google Scholar, 10Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (790) Google Scholar, 11Santoro B. Liu D.T. Yao H. Bartsch D. Kandel E.R. Siegelbaum S.A. Tibbs G.R. Cell. 1998; 93: 717-729Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). In the plasma membrane, HCN channel subunits assemble to homo- or heterotetrameric complexes, thereby generating a large variety of channel subtypes with distinct biophysical properties (12Frère S.G. Kuisle M. Lüthi A. Mol. Neurobiol. 2004; 30: 279-305Crossref PubMed Scopus (45) Google Scholar). Further complexity is probably generated in vivo by the interaction of HCN channels with auxiliary subunits (13Yu H. Wu J. Potapova I. Wymore R.T. Holmes B. Zuckerman J. Pan Z. Wang H. Shi W. Robinson R.B. El-Maghrabi M.R. Benjamin W. Dixon J. McKinnon D. Cohen I.S. Wymore R. Circ. Res. 2001; 88: E84-E87Crossref PubMed Google Scholar), interacting proteins (14Gravante B. Barbuti A. Milanesi R. Zappi I. Viscomi C. DiFrancesco D. J. Biol. Chem. 2004; 279: 43847-43853Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15Kimura K. Kitano J. Nakajima Y. Nakanishi S. Genes Cells. 2004; 9: 631-640Crossref PubMed Scopus (47) Google Scholar, 16Santoro B. Wainger B.J. Siegelbaum S.A. J. Neurosci. 2004; 24: 10750-10762Crossref PubMed Scopus (162) Google Scholar), and by post-translational modifications (e.g. N-linked glycosylation) (17Much B. Wahl-Schott C. Zong X. Schneider A. Baumann L. Moosmang S. Ludwig A. Biel M. J. Biol. Chem. 2003; 278: 43781-43786Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Whereas our knowledge of the structure and function of HCN channels has significantly increased over the last couple of years, there is only sparse information on the cellular regulation of these channels. It is well established that hormones and neurotransmitters can modulate Ih activity via G-protein pathways that modulate the cAMP concentration (1Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (985) Google Scholar). Cyclic AMP enhances channel activity by direct binding to a cyclic nucleotide binding domain (CNBD) present in the C terminus of HCN channels. The region linking the last membrane-spanning domain (S6) to the CNBD (the C-linker) has been shown to play a key role in coupling cAMP binding with channel opening (18Wainger B.J. DeGennaro M. Santoro B. Siegelbaum S.A. Tibbs G.R. Nature. 2001; 411: 805-810Crossref PubMed Scopus (403) Google Scholar, 19Wang J. Chen S. Siegelbaum S.A. J. Gen. Physiol. 2001; 118: 237-250Crossref PubMed Scopus (118) Google Scholar, 20Zhou L. Olivier N.B. Yao H. Young E.C. Siegelbaum S.A. Neuron. 2004; 44: 823-834Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Unlike in many other ion channels the modulation by cAMP does not seem to involve protein kinase A-mediated serine/threonine phosphorylation (21DiFrancesco D. Tortora P. Nature. 1991; 351: 145-147Crossref PubMed Scopus (653) Google Scholar). By contrast, several experimental observations suggest that nonreceptor protein-tyrosine kinases (PTKs), especially members of the Src family, may regulate HCN channels (1Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (985) Google Scholar). The HCN1 channel was originally identified in a yeast two-hybrid screen using the SH3 domain of Src as bait. However, it was not reported whether this interaction also occurs in native tissue and whether Src influences channel activity (22Santoro B. Grant S.G. Bartsch D. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14815-14820Crossref PubMed Scopus (228) Google Scholar). Based on experiments with genistein, a broad spectrum PTK inhibitor, it was suggested that cardiac Ih is regulated by tyrosine phosphorylation (23Wu J.Y. Cohen I.S. Pflugers Arch. 1997; 434: 509-514Crossref PubMed Scopus (38) Google Scholar, 24Wu J.Y. Yu H. Cohen I.S. Biochim. Biophys. Acta. 2000; 463: 15-19Crossref Scopus (28) Google Scholar, 25Yu H.G. Lu Z. Pan Z. Cohen I.S. Pflugers Arch. 2004; 447: 392-400Crossref PubMed Scopus (49) Google Scholar). However, this finding was challenged by another group reporting that the genistein effect is caused through nonselective interactions with the channel molecule (26Shibata S. Ono K. Iijima T. Br. J. Pharmacol. 1999; 128: 1284-1290Crossref PubMed Scopus (21) Google Scholar). Src is widely expressed in neurons and heart cells and has been shown to be an important regulator of voltage- and ligand-gated ion channels (27Davis M.J. Wu X. Nurkiewicz T.R. Kawasaki J. Gui P. Hill M.A. Wilson E. Am. J. Physiol. Heart. Circ. Physiol. 2001; 281: H1835-H1862Crossref PubMed Google Scholar). We therefore asked in the present study whether or not this kinase is involved in HCN channel modulation. Using genetic, biochemical, and electrophysiological methods, we demonstrate that Src specifically binds to the C terminus of HCN channels, phosphorylates the channels and thereby affects the activation kinetics of Ih. Moreover we identify a specific tyrosine residue in the C-linker region of HCN channels that is the molecular target of Src. Yeast Two-hybrid (YTH) Assay—C-terminal portions of murine HCN2 (mHCN2) were subcloned into plasmid pEG202 (Clontech) in-frame with the LexA DNA binding domain to yield the following bait proteins (see also Fig. 4A): CT, residues 449-863; L, residues 449-522; BD, residues 523-646; L+BD, residues 449-646; dC, residues 649-863; L-β8, residues 449-607; C′-BD, residues 487-646. The cDNAs of full-length chicken c-Src (residues 1-533) and the SH3 domain (residues 81-139) of c-Src were fused to B42 activation domain of the plasmid pJG4-5 (Clontech). Bacterial GST Fusion Proteins—C-terminal fragments of mHCN2 were subcloned into the EcoRI/BamHI site of pGEX2T (Amersham Biosciences) to generate the following GST-tagged proteins: CT, residues 448-863; L, residues 448-520; BD, residues 521-644; L+BD, residues 448-644; dC, residues 645-863. The SH3 domain of c-Src was cloned into the EcoRI/XhoI site of plasmid pET41a (Novagen) to produce a GST-SH3 fusion protein. Expression in HEK293 Cells—The cDNAs of wild-type and mutant mHCN2 and hHCN4 were subcloned into the mammalian expression vector pcDNA3 (Invitrogen). A Myc-tagged c-Src was constructed in the pcDNA3.1/Myc-His vector (Invitrogen). The dominant-negative mutant of chicken c-Src (Src-K295M) was subcloned in the pIRES2-EGFP vector (BD Biosciences Clontech). Mutant HCN channels were generated by standard PCR techniques. All plasmid constructs were verified by automated DNA sequencing. Yeast strain Saccharomyces cerevisiae EGY48 was used for YTH assays. pEG202 and pJG4-5 fusion plasmids, together with the lacZ reporter plasmid pSH18-34 (BD Biosciences, Clontech) were transformed into yeast by the lithium acetate method. To identify specific interactions transformants were grown on four different selective plates: 1) X-gal plates lacking uracil, histidine, and tryptophan, and containing galactose and raffinose as carbon source; 2) X-gal plates lacking uracil, histidine, and tryptophan, and containing glucose as carbon source; 3) plates lacking uracil, histidine, tryptophan, and leucine, and containing galactose and raffinose as carbon source; 4) plates lacking uracil, histidine, tryptophan, and leucine, and containing glucose as carbon source. An interaction was considered to be specific if a given transformant turned blue on plate condition 1, stayed white on plate condition 2, was able to grow on plate condition 3, and did not show growth on plate condition 4. Fig. 4B shows examples from selective plates 1 and 2. GST fusion proteins were expressed in the protease-deficient BL21 (DE3) strain of Escherichia coli by induction with 0.1 mm isopropyl-β-thiogalactopyranoside (IPTG), for 1 h at 37 °C. Bacteria were pelleted by centrifugation (10 min, 4 °C, 5,000 × g) and resuspended in 4 ml of PBS supplemented with protease inhibitor mix (PI) containing 1 μg/ml leupeptin, 1 μm pepstatin A, 1 μg/ml antipain, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm orthophenanthroline, 1 mm benzamidine, 1 mm iodacetamide. After a freeze-thaw cycle, lysozyme was added to a final concentration of 5 mg/ml. After incubation for 30 min on ice, 1% Triton X-100 was added, and the cell suspension was shaken for 30 min to improve solubility of the fusion proteins. Cell debris was pelleted by centrifugation (30 min, 10,000 × g, 4 °C). The supernatant was aliquoted and stored at -80 °C. For purification of GST fusion proteins, supernatant was incubated with 1 volume of 50% slurry glutathione-Sepharose beads (Amersham Biosciences) at 4 °C overnight. The loaded beads were pelleted by centrifugation (5 min, 500 × g, 4 °C) and washed twice with ice-cold PBS supplemented with PI. Beads were resuspended in 1 volume of PBS/PI and stored at 4 °C. About 3 μg of purified GST fusion protein was diluted into 1 ml of cell lysis buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 5 mm EDTA (pH 8.0), 1% Triton X-100). 10 μl of glutathione-Sepharose beads were added, and the mixture was incubated for 3 h at 4 °C. Beads were centrifuged at 10,000 × g for 2 min and washed three times with 1× HNTG buffer (50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerin, 5 mm EDTA (pH 8.0), 0.1% Triton X-100). After removing the supernatant, beads were incubated with total lysates from transfected or untransfected HEK293 cells for 4 h at 4 °C. The beads were pelleted after repeating the steps of centrifugation and washing. 20 μl of SDS loading buffer were added, and the mixture was heated 5 min at 100 °C before loading onto an SDS-PAGE gel. Beads preloaded with about 2 μg of GST fusion proteins (see GST pull-down assay) were washed three times with 1× HNTG buffer and once with kinase buffer (20 mm HEPES (pH 7.5), 10 mm MgCl2, 1 mm dithiothreitol, 200 μm pervanadate, 0.35 mm ATP). The beads then were mixed with 40 μl of reaction buffer (kinase buffer supplemented with 0.05 mg/ml bovine serum albumin and 0.1% β-mercaptoethanol) with or without 1 unit of purified c-Src (Oncogene Research Products). The reaction was carried out by shaking at room temperature for 15 min. After centrifugation and discarding the supernatant, the reaction was stopped by adding SDS loading buffer. All samples were then subjected to SDS-PAGE and Western blotting. The monoclonal anti-phosphotyrosine antibody 4G10 (anti-pY) (Upstate Biotechnology) was used to detect tyrosine-phosphorylated proteins. Membrane Fractions—Freshly isolated mouse brain tissue was washed with PBS and homogenized in ice-cold MOPS lysis buffer (0.3 mm sucrose, 20 mm MOPS, 1 mm EDTA) supplemented with 200 μm pervanadate and PI, using a glass cylinder and a Teflon plunger. Homogenates were centrifuged for 10 min at 5,000 × g. The pellet was rehomogenized and spun once more for 10 min. The combined supernatants were then centrifuged (45 min, 4 °C, 100,000 × g). The pellet comprising the total membrane fraction was resuspended in MOPS lysis buffer supplemented with 200 μm pervanadate and stored at -80 °C. Cell Lysates—Three days after transfection, HEK293 cells were washed twice with PBS and lysed with 500 μl of cell lysis buffer supplemented with 200 μm pervanadate and PI. After 10 min on ice the lysed cells were scraped off the dish and transferred to a reaction tube. The lysate was centrifuged (15 min, 4 °C, 12,000 × g), and the supernatant was subject to coimmunoprecipitation or stored at -80 °C. Total brain membrane fractions or cell lysates of HEK293 cells were incubated overnight at 4 °C with 25 μl of protein A-Sepharose (Sigma) and a specific antibody directed against one of the two examined proteins and 500 μl of HNTG buffer supplemented with 200 μm pervanadate and PI. Beads were pelleted by centrifugation (15 min, 4 °C, 12,000 × g) and washed three times with cold HNTG buffer supplemented with 200 μm pervanadate. Interacting proteins were visualized after boiling for 5 min in Laemmli sample buffer by SDS-PAGE and Western blot analysis. The following antibodies used were: anti-HCN2 (Alomone), anti-Src (GD11, Upstate Biotechnology Inc.), anti-GST (Amersham Biosciences), anti-Myc (9E10); anti-phosphotyrosine (4G10 (anti-pY), Upstate Biotechnology Inc. and p-Tyr-100, Cell Signaling Technology). An antibody against the cyclic nucleotide-gated channel CNGB3 (28Michalakis S. Geiger H. Haverkamp S. Hofmann F. Gerstner A. Biel M. Invest. Ophthalmol. Vis. Sci. 2005; 46: 1516-1524Crossref PubMed Scopus (114) Google Scholar) was used as control. The GST fusion protein containing the whole C terminus of HCN2 (amino acids 448-863) was in vitro phosphorylated by Src. Thereafter, the protein was boiled in SDS-PAGE sample buffer, separated on a 10% Tris-glycine gel, and stained with Coomassie Blue. The piece of gel containing the HCN2 fusion protein was excised and in-gel-digested with trypsin according to standard procedures. Tryptic peptides were extracted with 5% formic acid/50% acetronitrile, dried, and stored at -20 °C until analysis by mass spectrometry. A surveyor liquid chromatography system (ThermoFinnigan, San Jose, CA), consisting of degasser, MS Pump, and autosampler, equipped with a C18 trap column (RP, 300 μm × 5 mm, Agilent Technologies) and PicoFrit™ column: 75 μm × 100 mm, 15-μm tip packed with a 5-μm Aquasil C18 (ThermoFinnigan, San Jose, CA) was used. The samples were loaded onto the column with an RP gradient of 2-98% B over 180 min. RP solvents were 0.1% formic acid in either water (A) or acetonitrile (B). The flow rate was 200 nl/min. A Finnigan LTQ linear ion trap mass spectrometer equipped with an ESI microspray source was used for MS/MS experiment with ion transfer capillary of 160 °C and NSI voltage of 1.8 kV. The mass spectrometer was set that one full MS scan was followed by ten MS/MS scans on the ten most intense ions from the MS spectrum. Spectra from each fraction were searched by SEQUEST algorithm against the mHCN2 sequence. In these searches, differential modifications of 80 daltons to tyrosine residues were selected. For all sequences reported here, spectra were manually validated and contained sufficient information to assign not only the sequence, but also the site of phosphorylation. All output results were combined using home made software to delete the redundant data. HEK293 cells (DMSZ, Braunschweig, Germany) were maintained in DMEM medium (Invitrogen, Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin, and incubated at 37 °C with 10% CO2. For transfection, HEK293 cells were seeded on 6-well plates (diameter 3.5 cm) at a density of 700,000 cells per well. After 6 h, cells were transfected with expression plasmid DNA (0.6 μg of each plasmid per well) using the FuGENE 6 transfection reagent (Roche Diagnostics). For electrophysiological measurements, transfected cells were detached using 0.05% trypsin/0.5 mm EDTA (Invitrogen, Life Technologies, Inc.) and replated onto 12-mm poly-l-lysine-coated coverslips in 24-well plates. HL-1 cardiomyocytes were obtained from Dr. W. C. Claycomb (Lousiana State University Health Science Center, New Orleans, LA) and maintained in Claycomb Medium (JRH Biosciences, Andover, UK), supplemented with 10% fetal bovine serum, 4 mm l-glutamine, 10 μm noradrenaline, and penicillin-streptomycin as previously described (29Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1250) Google Scholar). Single HL-1 cells were detached from confluent cultures using trypsin-EDTA. Isolated cells were either replated or directly used for patch-clamp experiments. Dorsal root ganglion (DRG) neurons were isolated from adult mice as described by Wu et al. (30Wu Z.Z. Pan H.L. Brain. Res. 2004; 1029: 251-258Crossref PubMed Scopus (32) Google Scholar). Briefly, thoracic and lumbar DRGs were dissected and transferred immediately into DMEM medium. After removal of attached nerves and connective tissues ganglion fragments were placed in a microtube containing 1 ml of DMEM supplemented with trypsin (type I, 0.5 mg/ml, Sigma), collagenase (type I, 1 mg/ml, Sigma) and DNase (type I, 0.1 mg/ml, Sigma) and were incubated at 34 °C for 30 min. Thereafter, soybean trypsin inhibitor (type II-S, 1.25 mg/ml, Sigma) was added to stop trypsin digestion. The cell suspension was centrifuged (500 rpm, 3 min) to remove the supernatant and replenished with DMEM. Cells were then plated onto a 35-mm culture dish precoated with poly-l-lysine and kept in an incubator (37 °C, 10% CO2) for at least 1 h before electrophysiological recordings. Medium sized DRG neurons (30-45 μm) were selected for recordings. Pacemaker cells were prepared from sino-atrial node of adult mice as described by us previously (31Wahl-Schott C. Baumann L. Zong X. Biel M. J. Biol. Chem. 2005; 280: 13694-13700Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Currents of heterologously expressed HCN channels were measured at room temperature 2-3 days after transfection using whole cell voltage clamp technique. The extracellular solution was composed of (in mm): 110 NaCl, 0.5 MgCl2, 1.8 CaCl2, 5 HEPES, 30 KCl, pH 7.4 adjusted with NaOH. The intracellular solution contained (in mm): 130 KCl, 10 NaCl, 0.5 MgCl2, 1 EGTA, 5 HEPES, pH 7.4 adjusted with KOH. For measurement of HCN currents of HL-1 cells the pipette solution was composed according to Sartiani et al. (32Sartiani L. Bochet P. Cerbai E. Mugelli A. Fischmeister R. J. Physiol. 2002; 545: 81-92Crossref PubMed Scopus (70) Google Scholar) (in mm): 120 potassium aspartate, 10 TEA-Cl, 0.4 Na2GTP, 2 MgCl2, 11 EGTA, 5 CaCl2, 10 HEPES, pH adjusted to 7.4 with KOH. The bath solution contained (in mm): 110 NaCl, 30 KCl, 1.8 CaCl2, 0.5 MgCl2, 2 BaCl2, 5 HEPES, pH adjusted to 7.4 with NaOH. For measurement of Ih currents of DRG neurons the pipette solution was composed (in mm): 130 potassium aspartate, 10 NaCl, 0.5 MgCl2, 1 EGTA, 1 CaCl2, 3 Mg-ATP, 5 HEPES, pH adjusted to 7.4 with KOH. The bath solution contained (mm): 110 NaCl, 30 KCl, 1.8 CaCl2, 0.5 MgCl2, 5 BaCl2, 5 HEPES, pH adjusted to 7.4 with NaOH. The Ih currents of sinoatrial node cells were measured as described previously (31Wahl-Schott C. Baumann L. Zong X. Biel M. J. Biol. Chem. 2005; 280: 13694-13700Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Pipettes were pulled from borosilicate glass capillaries (GC150TF, Harvard Apparatus LTD) and had resistances of 2-3 MΩ when filled with the intracellular solution. Src kinase inhibitors (PP2 and genistein) and their respective inactive derivatives (PP3, daidzein) were purchased from Calbiochem (Germany). Stock solutions of these substances were prepared in Me2SO and were freshly diluted at least 1:1,000 in bath solution before using in experiments. The effects of Src inhibitors were determined after incubation of cells with the substances for at least 10 min. Data were acquired at 10 kHz using an Axopatch 200B amplifier and pClamp 8 (Axon Instruments). Voltage clamp data were stored on the computer hard disk and analyzed off-line by using Clampfit 8 (Axon Instruments). Steady-state activation curves were determined by hyperpolarizing voltages of -140 mV to -30 mV from a holding potential of -40 mV for 3.2 s (for wild-type and mutant HCN2) and 4.8 s (for HCN4) followed by a step to -140 mV. Tail currents measured immediately after the final step to -140 mV, were normalized by the maximal current (Imax) and plotted as a function of the preceding membrane potential. The data points were fitted with the Boltzmann function: (I - Imin)/(Imax - Imin) = {1 - exp[(Vm - V0.5)/k]} where Imin is an offset caused by a nonzero holding current, Vm is the test potential, V0.5 is the membrane potential for half-maximal activation, and k is the slope factor. Time constants of channel activation (τact) of wild-type and mutant HCN2 and HCN4 channels were determined by monoexponential (or biexponential in the case of HCN2-Y476F and Ih of DRG neurons) function fitting the current evoked during hyperpolarizing voltage pulses to -140 mV unless otherwise specified. As has been described earlier (33Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (315) Google Scholar) the initial lag in the activation of HCN channels was excluded from the fitting procedure. Statistical analyses were performed with Origin6.1 (OriginLab). Data are presented as mean ± S.E. (n = number of recorded cells). Inhibition of Src Slows Down Kinetics of Expressed HCN2 Channel—We investigated the effect of Src on HCN2 channels expressed in HEK293 cells. These cells endogenously express substantial amounts of Src making them a suitable system for our purpose (Fig. 1A). Preincubation of HCN2-expressing cells with the Src kinase inhibitor PP2 (10 μm) led to a profound deceleration of HCN2 activation kinetics (Fig. 1, B and F). The activation time constant (τact) at -140 mV increased from 277 ± 14 ms (n = 13) at control conditions to 538 ± 29 ms (n = 19) in the presence of PP2. In contrast, PP2 had no effect on current densities (pA/pF: PP2, -78.9 ± 12.4 (n = 25); control, -84.4 ± 12.5 (n = 27), p > 0.05). The effect on τact was present over the whole voltage range (supplemental Fig. S1). By contrast, PP3, an inactive analogue of PP2, did not affect current kinetics (Fig. 1, C and F, supplemental Fig. S1). Similarly, another Src kinase inhibitor, genistein (Fig. 1, D and F), but not its inactive analogue daidzein (Fig. 1F), also slowed down the HCN2 current. The finding that two structurally unrelated PTK inhibitors affected HCN2 in the same fashion argued against a nonspecific action of these agents. More likely, the effect of PP2 and genistein was caused by specific inhibition of Src and subsequent channel dephosphorylation by cellular tyrosine phosphatases. To further strengthen this hypothesis, we cotransfected HEK293 cells with HCN2 and a catalytically inactive Src mutant, Src-K295M (Src-KM) (34Snyder M.A. Bishop J.M. McGrath J.P. Levinson A.D. Mol. Cell. Biol. 1985; 5: 1772-1779Crossref PubMed Scopus (82) Google Scholar). Indeed, HCN2 currents obtained from these cells activated with slower kinetics than control currents (τact = 353 ± 7.4 (n = 6); Fig. 1, E and F). The effect induced by Src-K295M was somewhat weaker than that of PP2, probably because of the high levels of endogenous wild-type Src. Inhibition of Src also led to inhibition of deactivation kinetics (Fig. 2, A and B). In contrast, neither PP2 nor PP3 did affect the voltage dependence of activation (Fig. 2, C and D). Regulation of HCN2 by cAMP was principally preserved in the presence of PP2. Cyclic AMP shifted the half-maximal activation voltage (V0.5) by about +10 mV and speeded up activation kinetics (TABLE ONE). Interestingly, however, the activation kinetics in the presence of saturating cAMP + PP2 was consistently slower than that observed with cAMP alone (τact(PP2, cAMP) = 272 ± 23 ms (n = 12); τact(cAMP) = 163 ± 6.84 ms (n = 8)) indicating that the decelerating effect of PP2 persisted in the presence of cAMP.TABLE ONEEffects of cAMP on HCN2 current in the absence and presence of PP2Channels0 mm cAMP1 mm cAMPV0.5knτactnV0.5nknτactnmVmVmsmVmVmsHCN2−94.2 ± 0.896.88 ± 0.3812250 ± 1410−84.6 ± 0.9487.91 ± 0.448164 ± 6.848HCN2 + PP2−95.6 ± 0.735.65 ± 0.298633 ± 258−85.4 ± 1.13117.35 ± 0.5311272 ± 2312 Open table in a new tab HCN2 Binds Src and Undergoes Tyrosine Phosphorylation—We performed a series of immunoprecipitation experiments to determine whether HCN2 is a substrate of Src (Fig. 3A). In the first set of experiments, cells expressing HCN2 were preincubated with the tyrosine phosphatase inhibitor pervanadate (PV) prior to immunoprecipitation with anti-HCN2. A specific band corresponding to the molecular mass of HCN2 (105 kDa) was detected with a specific anti-phosphotyrosine antibody (anti-pY). This band was significantly weaker when cells were pretreated with both PV and PP2. Similarly, the intensity of the band was reduced in HCN2/Src-KM-coexpressing cells pretreated with PV. Taken together, these findings suggested that HCN2 undergoes tyrosine phosphorylation by Src and is dephosphorylated by cellular phosphatases if Src is inhibited. We next studied whether an interaction between Src and HCN also occurs in native tissue. To this end, immunoprecipitations with mouse brain membrane fractions were performed (Fig. 3B). Src was present in immunoprecipitates obtained with anti-HCN2 but not with a control antibody (anti-CNGB3) indicating that Src is bound to HCN2 in vivo. Moreov" @default.
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