FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2153500492> ?p ?o ?g. }

• W2153500492 endingPage "6072" @default.
• W2153500492 startingPage "6069" @default.
• W2153500492 abstract "An important step toward understanding the molecular basis of the functional diversity of pacemaker currents in spontaneously active cells has been the identification of a gene family encoding hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN) channels. Three of the four gene products that have been expressed so far give rise to pacemaker channels with distinct activation kinetics and are differentially distributed among the brain, with considerable overlap between some isoforms. This raises the possibility that HCN channels may coassemble to form heteromeric channels in some areas, similar to other K+ channels. In this study, we have provided evidence for functional heteromerization of HCN1 and HCN2 channels using a concatenated cDNA construct encoding two connected subunits. We have observed that heteromeric channels activate several-fold faster than HCN2 and only a little slower than HCN1. Furthermore, the voltage dependence of activation is more similar to HCN2, whereas the cAMP sensitivity is intermediate between HCN1 and HCN2. This phenotype shows marked similarity to the current arising from coexpressed HCN1 and HCN2 subunits in oocytes and the native pacemaker current in CA1 pyramidal neurons. We suggest that heteromerization may increase the functional diversity beyond the levels expected from the number of HCN channel genes and their differential distribution. An important step toward understanding the molecular basis of the functional diversity of pacemaker currents in spontaneously active cells has been the identification of a gene family encoding hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN) channels. Three of the four gene products that have been expressed so far give rise to pacemaker channels with distinct activation kinetics and are differentially distributed among the brain, with considerable overlap between some isoforms. This raises the possibility that HCN channels may coassemble to form heteromeric channels in some areas, similar to other K+ channels. In this study, we have provided evidence for functional heteromerization of HCN1 and HCN2 channels using a concatenated cDNA construct encoding two connected subunits. We have observed that heteromeric channels activate several-fold faster than HCN2 and only a little slower than HCN1. Furthermore, the voltage dependence of activation is more similar to HCN2, whereas the cAMP sensitivity is intermediate between HCN1 and HCN2. This phenotype shows marked similarity to the current arising from coexpressed HCN1 and HCN2 subunits in oocytes and the native pacemaker current in CA1 pyramidal neurons. We suggest that heteromerization may increase the functional diversity beyond the levels expected from the number of HCN channel genes and their differential distribution. hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective copy RNA μ-opioid receptor 5-HT4(a) receptor The newly cloned hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN)1 ion channels give rise to currents that are almost indistinguishable or very similar to native pacemaker currents termed If in heart (1DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (659) Google Scholar) andIh or Iq in brain tissue (2Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (969) Google Scholar). To date, three of the four HCN genes have been functionally expressed, giving rise to pacemaker currents with distinct gating properties (for review see Refs. 3Clapham D.E. Neuron. 1998; 21: 5-7Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Santoro B. Tibbs G.R. Ann. N. Y. Acad. Sci. 1999; 868: 741-764Crossref PubMed Scopus (302) Google Scholar, 5Biel M. Ludwig A. Zong X. Hofmann F. Rev. Physiol. Biochem. Pharmacol. 1999; 136: 165-181Crossref PubMed Google Scholar). HCN1 channels display fast gating properties and are almost unaffected by cAMP (6Santoro 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 (576) Google Scholar), similar to Ih recorded from some hippocampal pyramidal cells. HCN2 channels activate slowly and are strongly modulated by cAMP (7Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (778) Google Scholar, 8Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (310) Google Scholar), similar to native pacemaker currents in some cardiac cells. HCN4 channels activate very slowly and also respond strongly to cAMP (9Seifert R. Scholten A. Gauss R. Mincheva A. Lichter P. Kaupp U.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9391-9396Crossref PubMed Scopus (228) Google Scholar, 10Ishii T.M. Takano M. Xie L.H. Noma A. Ohmori H. J. Biol. Chem. 1999; 274: 12835-12839Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), similar to Ih in thalamic relay neurons. Expression patterns have confirmed that HCN1 is expressed restrictively in neurons of the neocortex, hippocampus, cerebellar cortex, and brainstem nuclei (6Santoro 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 (576) Google Scholar, 11Santoro 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, 12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). In contrast, HCN2 is widely expressed throughout the brain with prominent labeling of thalamic and brainstem nuclei as well as in heart tissue (6Santoro 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 (576) Google Scholar, 7Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (778) Google Scholar). Finally, HCN4 is predominantly expressed in thalamus, heart, and testis (9Seifert R. Scholten A. Gauss R. Mincheva A. Lichter P. Kaupp U.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9391-9396Crossref PubMed Scopus (228) Google Scholar). This distinct distribution pattern of HCN isoforms across the brain has been suggested to attribute to functional heterogeneity of native neuronal pacemaker currents (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). Nevertheless, considerable overlap was observed between HCN1 and HCN2 using in situ hybridization, namely in hippocampal pyramidal cells and some brainstem nuclei (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). Moreover, the coexistence of HCN1 and HCN2 mRNA has been demonstrated at the single-cell level for hippocampal CA1 neurons (13Franz O. Liss B. Neu A. Roeper J. Eur J. Neurosci. 2000; 12: 2685-2693Crossref PubMed Scopus (109) Google Scholar), raising the possibility that heteromeric channel complexes could form in some areas. The functional heteromerization of K+ channels with a tetrameric subunit stoichiometry has previously been demonstrated for voltage-gated K+ channels (14Christie M.J. North R.A. Osborne P.B. Douglass J. Adelman J.P. Neuron. 1990; 4: 405-411Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 15Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Sewing S. Pongs O. Nature. 1990; 345: 535-537Crossref PubMed Scopus (340) Google Scholar, 16Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1990; 345: 530-534Crossref PubMed Scopus (377) Google Scholar, 17Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (966) Google Scholar, 18Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (288) Google Scholar, 19Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (512) Google Scholar) as well as inwardly rectifying K+ channels (20Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar, 21Pessia M. Tucker S.J. Lee K. Bond C.T. Adelman J.P. EMBO J. 1996; 15: 2980-2987Crossref PubMed Scopus (169) Google Scholar) but is currently unknown for HCN channels. HCN channel subunits have an overall structure similar to voltage-gated K+ channel subunits, including 6 transmembrane domains (S1–S6), a selectivity filter containing the GYG motif, and a putative voltage sensor (S4) containing a positively charged residue at every third position. Based on these structural similarities it has been suggested that HCN channels will most likely adopt the general tetrameric subunit architecture of both 6- and 2-transmembrane segment K+ channels (4Santoro B. Tibbs G.R. Ann. N. Y. Acad. Sci. 1999; 868: 741-764Crossref PubMed Scopus (302) Google Scholar). To investigate the possibility that functional heteromeric HCN channels may form by coassembly of different HCN isoforms, we expressed a concatenated cDNA construct encoding a covalently linked HCN1 and HCN2 subunit into Xenopus oocytes. Currents were recorded using the 2-microelectrode voltage clamp technique and were compared with the biophysical properties of channels formed upon coexpression of HCN1 and HCN2 subunits and channels formed upon expression of HCN1 and HCN2 alone. The cAMP sensitivity of the homomeric and heteromeric HCN channels was assessed by coexpression of a G-protein-coupled receptor. The entire coding sequence for the mouse HCN1 and HCN2 channels (originally termed mBCNG-1 and mBCNG-2, respectively) were subcloned into the vector pSD64TF and pGEMHE, respectively. Plasmids were first linearized either with BamHI (for HCN1) or with SphI (for HCN2). Next, the cRNAs were synthesized from the linearized plasmids using the large scale SP6 (for HCN1) or T7 (for HCN2) mMessage mMachine transcription kit (Ambion). A concatenated construct encoding a covalently linked HCN1 and HCN2 subunit was engineered by digesting HCN1 with PshAI (blunt) and SpeI. HCN2 was digested with SmaI (blunt) and SpeI and ligated into the corresponding sites of the previous construct. The ligation product, HCN1·HCN2, was linearized with XbaI for size verification on an agarose gel and in vitrotranscription using the large scale SP6 mMessage mMachine transcription kit. The encoded dimer lacks the last 45 residues of the HCN1 C terminus and contains a 9-amino acid linker (GDPNSPFLA) that connects HCN1-Gln865stop to HCN2-M1 (see Fig.1 A). The human μ-opioid receptor (MOR) cDNA was subcloned into our custom high expression vector, pGEMHE (17Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (966) Google Scholar) and linearized for transcription as previously described (22Ulens C. Van Boven M. Daenens P. Tytgat J. J. Pharmacol. Exp. Ther. 2000; 294: 1024-1033PubMed Google Scholar). The rat GαsQ227L cDNA clone (23Chan J.S. Chiu T.T. Wong Y.H. J. Neurochem. 1995; 65: 2682-2689Crossref PubMed Scopus (86) Google Scholar) was subcloned in pcDNA1.1 and linearized with XbaI for in vitro transcription. The mouse 5-HT4(a)R cDNA clone (24Claeysen S. Sebben M. Becamel C. Bockaert J. Dumuis A. Mol. Pharmacol. 1999; 55: 910-920PubMed Google Scholar) in its original vector (pRK5) was first subcloned into theXbaI and HindIII sites of pSGEM, a modified version of pGEMHE (25Villmann C. Bull L. Hollmann M. J. Neurosci. 1997; 17: 7634-7643Crossref PubMed Google Scholar). For in vitro transcription, 5-HT4(a)R/pSGEM, was linearized with NheI. The capped cRNAs were synthesized from the linearized plasmids using the large-scale T7 mMessage mMachine transcription kit. The isolation of Xenopus laevis oocytes was as previously described (17Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (966) Google Scholar). Oocytes were injected with 10 ng/50 nl HCN1, HCN2, or HCN1·HCN2 cRNA. For determination of cAMP sensitivity, oocytes were coinjected with 0.05 ng of MOR; 0.05 ng of GαsQ227L or 0.01 ng of 5-HT4(a)R; and 10 ng of HCN1, HCN2, or HCN1·HCN2 (final injection volume, 50 nl). Injected oocytes were maintained in ND-96 solution (KCl 2 mm, NaCl 96 mm, MgCl2 1 mm, CaCl21.8 mm, HEPES 5 mm, pH 7.5) supplemented with 50 μg/ml gentamicin sulfate. Whole-cell currents from oocytes were recorded as previously described (22Ulens C. Van Boven M. Daenens P. Tytgat J. J. Pharmacol. Exp. Ther. 2000; 294: 1024-1033PubMed Google Scholar). Experiments were carried out using a high potassium (HK) external solution (KCl 96 mm, NaCl 2 mm, MgCl2 1 mm, CaCl2 1.8 mm, HEPES 5 mm, pH 7.5). Reversal potentials were determined in both HK and low potassium (ND-96) external solution (NaCl 96 mm, KCl 2 mm, MgCl2 1 mm, CaCl21.8 mm, HEPES 5 mm, pH 7.5). Cesium sensitivity of all the channel constructs was assessed by application of a hyperpolarizing pulse to −100 mV, long enough to achieve a fully activated state (4 s for HCN1 channels, 15 s for HCN2 channels, and 4 s for tandem HCN1·HCN2 channels). In this study, we coexpressed either μ-opioid or 5-HT4(a) receptors with HCN channels. Both receptors are representative members of the G-protein-coupled receptor superfamily that display an overlapping distribution pattern with HCN1 and HCN2 isoforms in the brain (24Claeysen S. Sebben M. Becamel C. Bockaert J. Dumuis A. Mol. Pharmacol. 1999; 55: 910-920PubMed Google Scholar, 26Peckys D. Landwehrmeyer G.B. Neuroscience. 1999; 88: 1093-1135Crossref PubMed Scopus (201) Google Scholar) and activate adenylyl cyclase (23Chan J.S. Chiu T.T. Wong Y.H. J. Neurochem. 1995; 65: 2682-2689Crossref PubMed Scopus (86) Google Scholar, 24Claeysen S. Sebben M. Becamel C. Bockaert J. Dumuis A. Mol. Pharmacol. 1999; 55: 910-920PubMed Google Scholar). The pathway through which the effect occurs involves Gβγ activation of adenylyl cyclase (27Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (741) Google Scholar, 28Federman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (479) Google Scholar) and requires coexpression of GαsQ227L (a constitutively active mutant) in the case of the μ-opioid receptor (23Chan J.S. Chiu T.T. Wong Y.H. J. Neurochem. 1995; 65: 2682-2689Crossref PubMed Scopus (86) Google Scholar). The effect of the 5-HT4(a) receptor, in part caused by its constitutive activity (24Claeysen S. Sebben M. Becamel C. Bockaert J. Dumuis A. Mol. Pharmacol. 1999; 55: 910-920PubMed Google Scholar), occurs directly through Gαsactivation. HCN currents were recorded before, during, and after application of an agonist (1 μm DAMGO for μ-opioid receptors and 1 μm 5-HT for 5-HT4(a)receptors) to activate the receptor. Current-voltage relationships were obtained by measuring the current amplitude after application of 4 s hyperpolarizing test pulses and normalization to the fully activated current at −110 mV (HCN1), −115 mV (HCN1·HCN2 tandem), and −125 mV (HCN2). Boltzmann activation curves were constructed using peak tail current amplitudes observed upon application of subsequent hyperpolarizing test pulses. Current values were plotted as a function of the applied voltage step and fitted with a Boltzmann equation, I=Ioff+Imax1+exp(−(Vtest−V1/2)/S)Equation 1 where I represents the current,Ioff the offset, Imax the maximal current, Vtest the applied test voltage,V 12 the midpoint potential, and S the slope of the Boltzmann curve. Curves of n experiments were normalized to the maximal tail current amplitude and corrected for the offset. Time constants of activation were determined by fitting the late rising phase of the current with a double exponential function. The initial lag of the rising phase was excluded from the fitting procedure to obtain an appropriate fit. The lag phase maximally comprised the first 12 ms, 32 ms, and 280 ms of the rising current for HCN1, HCN1·HCN2 dimers, and HCN2, respectively. Averaged data are indicated as mean ± S.E. Statistical analysis of differences between groups was carried out with Student's t test, and a probability of 0.05 was taken as the level of statistical significance. In this study, we investigated the possible heteromerization of HCN channels by expressing a concatenated cDNA construct encoding a covalently linked HCN1 and HCN2 subunit (Fig.1 A) into Xenopusoocytes. Both HCN1 and HCN2 are members of the HCN gene family (3Clapham D.E. Neuron. 1998; 21: 5-7Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 5Biel M. Ludwig A. Zong X. Hofmann F. Rev. Physiol. Biochem. Pharmacol. 1999; 136: 165-181Crossref PubMed Google Scholar,6Santoro 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 (576) Google Scholar), encoding pacemaker channels with easily distinguishable kinetic properties. For comparison, all records shown in Fig. 1 Bwere evoked using the same voltage step protocol. Homomeric HCN1 channels activate relatively fast, whereas homomeric HCN2 channels activate more slowly (Fig. 1 B, TableI). Expression of cRNA encoding the dimeric construct gives rise to currents that activate somewhat slower than HCN1, but still several-fold faster than HCN2 (Fig. 1 B, Table I). This finding provides important evidence that the concatenated construct encodes heteromeric HCN1·HCN2 channels, because the unexpectedly fast kinetics cannot be accounted for by the mathematical average expected from homomeric HCN1 and HCN2 channels. Furthermore, the heteromeric phenotype shows marked resemblance to currents that were evoked from oocytes coinjected with equal amounts of HCN1 and HCN2 cRNA (Fig. 1 B). This result suggests that HCN1 and HCN2 subunits could spontaneously coassemble to form a population of channels with a subunit arrangement similar to the heteromeric channels formed by HCN1·HCN2 dimers.Table IComparison between HCN clones and Ih in CA1 hippocampal neuronscAMP-dependent shift of V 12 , S (2-s pulses)mVmsmVHCN10.02 ± 1.13 1-aDetermined with a coexpressed μ-opioid receptor and GαSQ227L.23.7 ± 2.9−66.6 ± 0.10.3 ± 1.4 1-bDetermined with a coexpressed 5-HT4(a) receptor.138 ± 11−13.2 ± 0.06HCN215.4 ± 1.0 1-aDetermined with a coexpressed μ-opioid receptor and GαSQ227L.287.2 ± 8.9−91.4 ± 1.614.4 ± 1.1 1-bDetermined with a coexpressed 5-HT4(a) receptor.1794 ± 98−7.9 ± 0.8HCN1 · HCN27.1 ± 1.3 1-aDetermined with a coexpressed μ-opioid receptor and GαSQ227L.99.8 ± 7.7−80.8 ± 1.97.3 ± 1.3 1-bDetermined with a coexpressed 5-HT4(a) receptor.440 ± 16−11.6 ± 1.1Ih(13)7 1-cDetermined by including 1 mm 8-bromo-cAMP in the pipette solution.64.4 ± 2.9−84.3 ± 0.5537 ± 47−10.1 ± 0.31-a Determined with a coexpressed μ-opioid receptor and GαSQ227L.1-b Determined with a coexpressed 5-HT4(a) receptor.1-c Determined by including 1 mm 8-bromo-cAMP in the pipette solution. Open table in a new tab Current-voltage relationships were constructed from the records in Fig.1 B and are compared in Fig. 1 C. It should be noted that the 4-s test pulses applied to record the traces shown in Fig. 1 B do not allow HCN2 channels to obtain a steady-state level of activation because of their relatively slow time course of activation (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). To make a valid comparison of the voltage dependence of activation between homomeric and heteromeric HCN1 and HCN2 channels, we calculated half-maximal values of activation (V 12 ) from experiments with voltage steps of increasing duration (Fig. 1 D). These results show that the use of relatively short hyperpolarizing pulses shifts the estimates of the V 12 -values to more negative potentials. Comparison of calculated V 12 -values shows that heteromeric HCN1·HCN2 channels activate at membrane potentials intermediate to homomeric HCN1 and HCN2 channels for pulses ≤5 s. However, heteromeric HCN1·HCN2 channels activate at membrane potentials more negative than the average of steady-state V 12 -values for homomeric HCN1 and HCN2 channels (average indicated with a dashed line), for pulses long enough to achieve steady-state levels of activation (>10 s). This result would suggest that a voltage-dependent transition in the opening of the heteromeric HCN1·HCN2 channel complex is limited by the isoform that activates at the most negative membrane voltages, as in the case of HCN2. As could be observed from the current traces in Fig. 1 B, heteromeric HCN1·HCN2 channels activate with a faster time course than the average of homomeric HCN1 and HCN2 channels predicts. For clarification, we have superimposed traces from homomeric and heteromeric channels recorded during a hyperpolarizing step to −90 mV (Fig. 2 A). The current trace recorded from oocytes coexpressing HCN1 and HCN2 channels perfectly matches with the trace obtained from the tandem HCN1·HCN2 channels, suggesting that channels are formed with a subunit stoichiometry similar to the defined subunit arrangement of the tandem-linked channels. Next, we quantified the kinetics of activation by fitting current traces during hyperpolarizing voltage steps (as shown in Fig.1 B) with two exponential functions (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). The calculated fast and slow time constants of activation were plotted as a function of the applied test potential and are shown in Fig. 2, B andC, respectively. Values calculated for the traces in Fig.2 A were τfast = 214 ± 20 ms (n = 6) and τslow = 909 ± 54 ms (n = 6) for tandem HCN1·HCN2 channels and τfast = 168 ± 8 ms (n = 6) and τslow = 944 ± 49 ms (n = 6) for coexpressed HCN1 and HCN2 channels. Homomeric HCN1 and HCN2 channels as well as heteromeric HCN1·HCN2 channels have slow time constants of activation that are 5- to 10-fold higher than fast time constants. Compared with the respective time constants for homomeric HCN1 channels, homomeric HCN2 channels have fast and slow time constants that are ∼10-fold higher. These results are in accordance with recently reported data on pacemaker channels in the mouse CNS (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). Heteromeric HCN1·HCN2 channels have time constants of activation that are significantly faster than the average of homomeric HCN1 and HCN2 channels predicts (indicated with a dashed line). Heteromeric HCN1·HCN2 channels activate similarly to homomeric HCN1 channels, with fast and slow time constants of activation that are only about 3-fold higher. This result suggests that HCN1 subunits dominate the activation rate of the heteromeric channel complex. Another striking feature that discriminates between homomeric HCN1 and HCN2 channels is their differential sensitivity to cAMP (6Santoro 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 (576) Google Scholar, 7Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (778) Google Scholar). Hormones and neurotransmitters acting through the second messenger cAMP finely tune the activation of the pacemaker current by shifting the voltage dependence of activation along the voltage axis (1DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (659) Google Scholar, 2Pape H.C. Annu. Rev. Physiol. 1996; 58: 299-327Crossref PubMed Scopus (969) Google Scholar). The underlying mechanism involves direct binding of cAMP to the channel cyclic nucleotide binding domain (29DiFrancesco D. Tortora P. Nature. 1991; 351: 145-147Crossref PubMed Scopus (636) Google Scholar). Similar to studies using excised inside-out patches (6Santoro 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 (576) Google Scholar, 7Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (778) Google Scholar), we found that activation of a coexpressed G-protein-coupled receptor, either the 5-HT4(a) or μ-opioid receptor, shifts the V 12 -value by 14.9 mV toward more positive potentials for homomeric HCN2 channels and no shift for homomeric HCN1 channels (Fig.3 A). Intriguingly, heteromeric HCN1·HCN2 channels display a cAMP sensitivity that is intermediate to homomeric HCN1 and HCN2 channels, with a 7.2 mV shift toward more positive potentials in response to activation of coexpressed G-protein-coupled receptor. Representative current traces from experiments using homomeric and heteromeric channels coexpressed with μ-opioid or 5-HT4(a) receptors are shown in Fig. 3,B and C, respectively. Based on the structural similarities of the 6 transmembrane domains, the pore-forming region and cytoplasmic N and C termini of HCN channels with other K+ channels, it has previously been suggested that HCN channels will most likely conform to the tetrameric subunit arrangement of both the 6- and 2-transmembrane domain K+ channels (4Santoro B. Tibbs G.R. Ann. N. Y. Acad. Sci. 1999; 868: 741-764Crossref PubMed Scopus (302) Google Scholar). With respect to our results with HCN1·HCN2 tandems, this idea would suggest that functional channels are likely to be formed by coassembly of two HCN1·HCN2 tandems, adopting a HCN1/HCN2/HCN1/HCN2 or HCN1/HCN2/HCN2/HCN1 subunit stoichiometry. Our experimental results on the cAMP sensitivity of heteromeric HCN1·HCN2 channels support the idea of a tetrameric subunit arrangement, because the shift of theV 12 -value for heteromeric HCN1·HCN2 channels (7.2 mV) is exactly half of the shifts determined for homomeric HCN1 (0.2 mV) and HCN2 (14.9 mV) channels. Finally, HCN channels are characterized by weak selectivity for K+ over Na+ and the inward current is sensitive to block by application of external Cs+. Therefore, we compared the reversal potentials and the cesium sensitivity for homomeric HCN1 and HCN2 channels with heteromeric HCN1·HCN2 channels. Reversal potentials determined in ND-96 solution (2 mmK+) and HK solution (96 mm K+) were −30 ± 1 mV (n = 5) and −2 ± 2 mV (n = 6) for HCN1 channels, −29 ± 2 mV (n = 5) and 0 ± 1 mV (n = 6) for HCN2 channels, and −30 ± 2 mV (n = 6) and −1 ± 2 mV (n = 6) for tandem HCN1·HCN2 channels. Application of external Cs+ caused a complete inhibition of the inward current through HCN channels, characterized by IC50 values of 124 ± 28 μm(n = 6) for HCN1 channels, 63 ± 12 (n = 6) for HCN2 channels and 79 ± 11 (n = 6) for tandem HCN1·HCN2 channels. These results indicate that heteromeric HCN1·HCN2 channels share identical ion selectivity and cesium sensitivity with homomeric HCN1 and HCN2 channels. Taken together, our data raise the question about the relevance to native pacemaker currents. To address this issue, it is important to note that HCN isoforms not only differ in their biophysical properties and cAMP sensitivity, but also in their distinct distribution pattern across the brain (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar, 13Franz O. Liss B. Neu A. Roeper J. Eur J. Neurosci. 2000; 12: 2685-2693Crossref PubMed Scopus (109) Google Scholar, 30Moosmang S. Biel M. Hofmann F. Ludwig A. Biol. Chem. 1999; 380: 975-980Crossref PubMed Scopus (198) Google Scholar). Nevertheless, considerable overlap was shown between HCN1 and HCN2 using in situ hybridization, namely in hippocampal pyramidal cells and some brainstem nuclei (12Santoro B. Chen S. Luthi A. Pavlidis P. Shumyatsky G.P. Tibbs G.R. Siegelbaum S.A. J. Neurosci. 2000; 20: 5264-5275Crossref PubMed Google Scholar). Moreover, the coexistence of HCN1 and HCN2 mRNA has been demonstrated at the single cell level for hippocampal CA1 neurons (13Franz O. Liss B. Neu A. Roeper J. Eur J. Neurosci. 2000; 12: 2685-2693Crossref PubMed Scopus (109) Google Scholar), raising the possibility that heteromeric channel complexes could form in some areas. In Table I, we have compared our data to the results obtained by Franz et al. (13Franz O. Liss B. Neu A. Roeper J. Eur J. Neurosci. 2000; 12: 2685-2693Crossref PubMed Scopus (109) Google Scholar) based on the similar recording conditions (room temperature, whole cell configuration) and data analysis (double exponential fits to the time course of activation). The cAMP-induced shift of theV 12 -value for Ih in hippocampal CA1 neurons correlates well with the shift that we observed for heteromeric HCN1·HCN2 channels. This result can be expected from a population of channels that either consists of equal amounts of homomeric HCN1 and HCN2 channels or a population of channels with a heteromeric stoichiometry, similar to the tandem-linked channels. Comparison of the kinetic properties and the voltage dependence of activation for Ih in hippocampal CA1 neurons with our results however favors the formation of heteromeric channels, because equally distributed homomeric channels cannot account for the fast gating properties of the native Ih. In conclusion, the cAMP dependence and gating properties of tandem-linked HCN1 and HCN2 subunits reveal that HCN channels can function as heteromers, most likely tetramers. Based on the resemblance of the tandem it's distinct current phenotype with currents obtained from coexpressed HCN1 and HCN2 subunits and native pacemaker current in neurons in which different isoforms coexist, our results suggest that heteromerization of different HCN channels may further contribute to the functional heterogeneity of native pacemaker currents. Finally, the dimeric construct may also serve as a model to elucidate the physiological role of native pacemaker currents and as a relevant pharmacological tool to develop new compounds for these channels. We are very grateful to Dr. Steven A. Siegelbaum for his generous gift of mBCNG-1 and mBCNG-2 cDNAs and for sharing his comments on our work. The μ-opioid receptor clone was kindly provided by Dr. Lei Yu. The GαsQ227L clone was a gift from Dr. Yung H. Wong. The 5-HT4(a) receptor cDNA was kindly donated by Dr. S. Claeysen and Dr. A. Dumuis." @default.
• W2153500492 created "2016-06-24" @default.
• W2153500492 creator A5021345022 @default.
• W2153500492 creator A5050669467 @default.
• W2153500492 date "2001-03-01" @default.
• W2153500492 modified "2023-10-16" @default.
• W2153500492 title "Functional Heteromerization of HCN1 and HCN2 Pacemaker Channels" @default.
• W2153500492 cites W1507218771 @default.
• W2153500492 cites W1604214492 @default.
• W2153500492 cites W1893208393 @default.
• W2153500492 cites W1969583788 @default.
• W2153500492 cites W1974273055 @default.
• W2153500492 cites W1979103094 @default.
• W2153500492 cites W1979411530 @default.
• W2153500492 cites W1984286247 @default.
• W2153500492 cites W1992473638 @default.
• W2153500492 cites W1996071311 @default.
• W2153500492 cites W2004855428 @default.
• W2153500492 cites W2008142951 @default.
• W2153500492 cites W2010973571 @default.
• W2153500492 cites W2014880992 @default.
• W2153500492 cites W2018527368 @default.
• W2153500492 cites W2038894306 @default.
• W2153500492 cites W2053967009 @default.
• W2153500492 cites W2058606373 @default.
• W2153500492 cites W2058724065 @default.
• W2153500492 cites W2079852966 @default.
• W2153500492 cites W2084471888 @default.
• W2153500492 cites W2094529776 @default.
• W2153500492 cites W2101625991 @default.
• W2153500492 cites W2112196515 @default.
• W2153500492 cites W2162430687 @default.
• W2153500492 cites W2165340969 @default.
• W2153500492 cites W2171569887 @default.
• W2153500492 cites W2173794755 @default.
• W2153500492 doi "https://doi.org/10.1074/jbc.c000738200" @default.
• W2153500492 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11133998" @default.
• W2153500492 hasPublicationYear "2001" @default.
• W2153500492 type Work @default.
• W2153500492 sameAs 2153500492 @default.
• W2153500492 citedByCount "196" @default.
• W2153500492 countsByYear W21535004922012 @default.
• W2153500492 countsByYear W21535004922013 @default.
• W2153500492 countsByYear W21535004922014 @default.
• W2153500492 countsByYear W21535004922015 @default.
• W2153500492 countsByYear W21535004922016 @default.
• W2153500492 countsByYear W21535004922017 @default.
• W2153500492 countsByYear W21535004922018 @default.
• W2153500492 countsByYear W21535004922019 @default.
• W2153500492 countsByYear W21535004922020 @default.
• W2153500492 countsByYear W21535004922021 @default.
• W2153500492 countsByYear W21535004922022 @default.
• W2153500492 countsByYear W21535004922023 @default.
• W2153500492 crossrefType "journal-article" @default.
• W2153500492 hasAuthorship W2153500492A5021345022 @default.
• W2153500492 hasAuthorship W2153500492A5050669467 @default.
• W2153500492 hasBestOaLocation W21535004921 @default.
• W2153500492 hasConcept C144133560 @default.
• W2153500492 hasConceptScore W2153500492C144133560 @default.
• W2153500492 hasIssue "9" @default.
• W2153500492 hasLocation W21535004921 @default.
• W2153500492 hasOpenAccess W2153500492 @default.
• W2153500492 hasPrimaryLocation W21535004921 @default.
• W2153500492 hasRelatedWork W1491839574 @default.
• W2153500492 hasRelatedWork W2011945577 @default.
• W2153500492 hasRelatedWork W2596173151 @default.
• W2153500492 hasRelatedWork W2624733068 @default.
• W2153500492 hasRelatedWork W2765153054 @default.
• W2153500492 hasRelatedWork W2931838652 @default.
• W2153500492 hasRelatedWork W3080576469 @default.
• W2153500492 hasRelatedWork W3168296622 @default.
• W2153500492 hasRelatedWork W4231340554 @default.
• W2153500492 hasRelatedWork W2051331738 @default.
• W2153500492 hasVolume "276" @default.
• W2153500492 isParatext "false" @default.
• W2153500492 isRetracted "false" @default.
• W2153500492 magId "2153500492" @default.
• W2153500492 workType "article" @default.