FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2094516452> ?p ?o ?g. }

• W2094516452 endingPage "27061" @default.
• W2094516452 startingPage "27056" @default.
• W2094516452 abstract "Hyperpolarization-activated cation channels of the HCN gene family are crucial for the regulation of cell excitability. Importantly, these channels play a pivotal role in the control of cardiac and neuronal pacemaker activity. Dysfunction of HCN channels has been associated with human diseases, including cardiac arrhythmia, epilepsy, and neuropathic pain. The properties of three HCN channel isoforms (HCN1, HCN2, and HCN4) have been extensively investigated. By contrast, due to the lack of an efficient heterologous expression system, the functional characteristics of HCN3 were by and large unknown so far. Here, we have used lentiviral gene transfer to overexpress HCN3 in HEK293T cells. HCN3 currents revealed slow activation and deactivation kinetics and were effectively blocked by extracellular Cs+ and the bradycardic agent ivabradine. Cyclic AMP and cGMP had no significant impact on activation kinetics but induced a 5-mV shift of the half-maximal activation voltage (V0.5) to more hyperpolarized potentials. A negative shift of V0.5 induced by cyclic nucleotides is an unprecedented feature within the HCN channel family. The expression of HCN3 in mouse brain was examined by Western blot analysis using a specific antibody. High levels of protein were detected in olfactory bulb and hypothalamus. In contrast, only very low expression was found in cortex. Using reverse transcriptase PCR transcripts of HCN3 were also detected in heart ventricle. In conclusion, the distinct expression pattern in conjunction with the unusual biophysical properties implies that HCN3 may play an unique role in the body. Hyperpolarization-activated cation channels of the HCN gene family are crucial for the regulation of cell excitability. Importantly, these channels play a pivotal role in the control of cardiac and neuronal pacemaker activity. Dysfunction of HCN channels has been associated with human diseases, including cardiac arrhythmia, epilepsy, and neuropathic pain. The properties of three HCN channel isoforms (HCN1, HCN2, and HCN4) have been extensively investigated. By contrast, due to the lack of an efficient heterologous expression system, the functional characteristics of HCN3 were by and large unknown so far. Here, we have used lentiviral gene transfer to overexpress HCN3 in HEK293T cells. HCN3 currents revealed slow activation and deactivation kinetics and were effectively blocked by extracellular Cs+ and the bradycardic agent ivabradine. Cyclic AMP and cGMP had no significant impact on activation kinetics but induced a 5-mV shift of the half-maximal activation voltage (V0.5) to more hyperpolarized potentials. A negative shift of V0.5 induced by cyclic nucleotides is an unprecedented feature within the HCN channel family. The expression of HCN3 in mouse brain was examined by Western blot analysis using a specific antibody. High levels of protein were detected in olfactory bulb and hypothalamus. In contrast, only very low expression was found in cortex. Using reverse transcriptase PCR transcripts of HCN3 were also detected in heart ventricle. In conclusion, the distinct expression pattern in conjunction with the unusual biophysical properties implies that HCN3 may play an unique role in the body. The hyperpolarization-activated cation current, termed Ih or If, is widely expressed in heart cells and neurons. The current is best known for its prime role in the generation of rhythmic activity in cardiac and neuronal pacemaker cells (1Robinson R.B. Siegelbaum S.A. Annu. Rev. Physiol. 2003; 65: 453-480Crossref PubMed Scopus (900) Google Scholar, 2DiFrancesco D. Annu. Rev. Physiol. 1993; 55: 455-472Crossref PubMed Scopus (663) Google Scholar). Ih is also present in several types of non-pacing neurons where it contributes to various physiological functions, including the setting of the resting membrane potential, synaptic transmission, and dendritic integration. Recent evidence suggests that Ih is involved in diseases making the channel a promising target for drug therapy. For example, the dysfunction of cardiac Ih was identified in patients suffering from sick sinus bradycardia (3Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (307) Google Scholar, 4Ueda 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 (194) Google Scholar). Furthermore, it was proposed that overexpression of Ih in heart ventricle is associated with cardiac hypertrophy (5Cerbai E. Sartiani L. DePaoli P. Pino R. Maccherini M. Bizzarri F. DiCiolla F. Davoli G. Sani G. Mugelli A. J. Mol. Cell Cardiol. 2001; 33: 441-448Abstract Full Text PDF PubMed Scopus (112) Google Scholar). Ivabradine (S-16257-2), a blocker of Ih, is currently considered as a novel drug in the therapy of tachycardic arrhythmia and angina pectoris (6Bois P. Bescond J. Renaudon B. Lenfant J. Br. J. Pharmacol. 1996; 118: 1051-1057Crossref PubMed Scopus (263) Google Scholar). Ih is also likely to participate in neurological diseases. In particular, there is accumulating evidence that the current is involved in epileptogenesis (7Poolos N.P. Epilepsy Curr. 2004; 4: 3-6Crossref PubMed Google Scholar, 8Ludwig A. Budde T. Stieber J. Moosmang S. Wahl C. Holthoff K. Langebartels A. Wotjak C. Munsch T. Zong X. Feil S. Feil R. Lancel M. Chien K.R. Konnerth A. Pape H.C. Biel M. Hofmann F. EMBO J. 2003; 22: 216-224Crossref PubMed Scopus (413) Google Scholar). Moreover, overexpression of Ih was observed in rat models of peripheral nerve injury suggesting a potential role of this current in driving neuropathic pain (9Chaplan S.R. Guo H.Q. Lee D.H. Luo L. Liu C. Kuei C. Velumian A.A. Butler M.P. Brown S.M. Dubin A.E. J. Neurosci. 2003; 23: 1169-1178Crossref PubMed Google Scholar).Ih is encoded by a family of four hyperpolarization-activated cyclic nucleotide-gated (HCN 1The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-gated; HEK, human embryonic kidney; RT-PCR, reverse transcriptase PCR; cAMP, cyclic adenosine monophosphate; V0.5, voltage of half-maximal activation; Imax, maximal current; Iinst, instantaneous current; τact, time constant of channel activation; Mw, molecular weight; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein. 1The abbreviations used are: HCN, hyperpolarization-activated cyclic nucleotide-gated; HEK, human embryonic kidney; RT-PCR, reverse transcriptase PCR; cAMP, cyclic adenosine monophosphate; V0.5, voltage of half-maximal activation; Imax, maximal current; Iinst, instantaneous current; τact, time constant of channel activation; Mw, molecular weight; IRES, internal ribosome entry site; EGFP, enhanced green fluorescence protein.1-4) channels (1Robinson R.B. Siegelbaum S.A. Annu. Rev. Physiol. 2003; 65: 453-480Crossref PubMed Scopus (900) Google Scholar, 10Kaupp U.B. Seifert R. Annu. Rev. Physiol. 2001; 63: 235-257Crossref PubMed Scopus (304) Google Scholar). HCN channels are members of the 6-transmembrane superfamily of cation channels (11Hofmann F. Biel M. Kaupp U.B. Pharmacol. Rev. 2003; 55: 587-589Crossref PubMed Scopus (25) Google Scholar). A structural hallmark of all HCN channels is a cyclic nucleotide-binding domain in the C terminus that confers sensitivity to cAMP (12Zagotta W.N. Olivier N.B. Black K.D. Young E.C. Olson R. Gouaux E. Nature. 2003; 425: 200-205Crossref PubMed Scopus (480) Google Scholar, 13Wainger B.J. DeGennaro M. Santoro B. Siegelbaum S.A. Tibbs G.R. Nature. 2001; 411: 805-810Crossref PubMed Scopus (389) Google Scholar). So far, the functional properties of three members of the HCN channel family (HCN1, HCN2, and HCN4) have been extensively studied using heterologous expression. Moreover, the specific physiological relevance of these channels has been defined using genetargeting approaches in mice (8Ludwig A. Budde T. Stieber J. Moosmang S. Wahl C. Holthoff K. Langebartels A. Wotjak C. Munsch T. Zong X. Feil S. Feil R. Lancel M. Chien K.R. Konnerth A. Pape H.C. Biel M. Hofmann F. EMBO J. 2003; 22: 216-224Crossref PubMed Scopus (413) Google Scholar, 14Nolan M.F. Malleret G. Lee K.H. Gibbs E. Dudman J.T. Santoro B. Yin D. Thompson R.F. Siegelbaum S.A. Kandel E.R. Morozov A. Cell. 2003; 115: 551-564Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 15Stieber J. Herrmann S. Feil S. Loster J. Feil R. Biel M. Hofmann F. Ludwig A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15235-15240Crossref PubMed Scopus (371) Google Scholar). By contrast, there is only sparse information on the properties of the HCN3 channel (9Chaplan S.R. Guo H.Q. Lee D.H. Luo L. Liu C. Kuei C. Velumian A.A. Butler M.P. Brown S.M. Dubin A.E. J. Neurosci. 2003; 23: 1169-1178Crossref PubMed Google Scholar, 16Moosmang S. Stieber J. Zong X. Biel M. Hofmann F. Ludwig A. Eur. J. Biochem. 2001; 268: 1646-1652Crossref PubMed Scopus (348) Google Scholar). In situ hybridization and immunocytochemistry indicated that this channel is expressed at low levels in rat and mouse brains (17Moosmang S. Biel M. Hofmann F. Ludwig A. Biol. Chem. 1999; 380: 975-980Crossref PubMed Scopus (199) Google Scholar, 18Monteggia L.M. Eisch A.J. Tang M.D. Kaczmarek L.K. Nestler E.J. Brain Res. Mol. Brain Res. 2000; 81: 129-139Crossref PubMed Scopus (181) Google Scholar, 19Notomi T. Shigemoto R. J. Comp. Neurol. 2004; 471: 241-276Crossref PubMed Scopus (427) Google Scholar). The biophysical and pharmacological properties of the channel are by and large unknown.In the present study we set out to address this important issue. We investigated the expression levels of murine HCN3 in mouse brain regions using Western blot analysis. Moreover, we achieved robust expression of the channel in HEK293T cells using lentiviral expression vectors. We show that HCN3 reveals some properties shared by other HCN channel types but is unique among these channels by being rather inhibited than activated by cyclic nucleotides.MATERIALS AND METHODSLentiviral HCN2 and HCN3 Expression Vectors—The mouse HCN3 (mHCN3) coding sequence was excised as a 2.7-kb HindIII-SpeI fragment from the plasmid mHCN3/pcDNA3 (16Moosmang S. Stieber J. Zong X. Biel M. Hofmann F. Ludwig A. Eur. J. Biochem. 2001; 268: 1646-1652Crossref PubMed Scopus (348) Google Scholar). The mHCN3 was cloned via XbaI and SpeI sites into pBluescript II KS plasmid containing the IRES-EGFP coding sequence (Clontech). Lentiviral plasmid LV-HCN3 was prepared by replacing the GFP coding sequence with the XbaI-SalI HCN3-IRES-EGFP fragment. A schematic representation of the HCN3 lentiviral expression vector is given in Fig. 2. The lentiviral HCN2 expression vector LV-HCN2 was constructed accordingly by cloning the coding region of mHCN2 (20Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (779) Google Scholar) into the LV vector. Recombinant lentivirus as well as lentiviral particles were generated as described previously (21Pfeifer A. Brandon E.P. Kootstra N. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11450-11455Crossref PubMed Scopus (187) Google Scholar).Cell Culture—HEK293T cells (DSMZ, Braunschweig, Germany) infected with lentiviruses as previously described (21Pfeifer A. Brandon E.P. Kootstra N. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11450-11455Crossref PubMed Scopus (187) Google Scholar) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and incubated at 37 °C with 10% CO2 in T25, gelatin-fibronectin-coated flasks (Sigma). For electrophysiological experiments, glass coverslips were coated in 24-well dishes with poly-l-lysine (Sigma), and 30,000 cells/well were seeded.RT-PCR—Total RNA was isolated using TRIzol (Invitrogen) and subsequently treated with DNase I (Roche Applied Science). First strand cDNA was synthesized from 5 μg of RNA with the Superscript II H--Kit (Invitrogen) using oligo(dT) primers. HCN3 was amplified from 0.5 μl of cDNA using following primers and conditions: 5′-GTCCGCCGGGGCCTGGAT-3′ (forward), 5′-CCTCCCACTGGTGTATGTAGC-3′ (reverse); 40 cycles at 60 °C. Amplicons were separated on 5% polyacrylamide gels, stained with ethidium bromide, and visualized on a Gel Doc 2000 system (Bio-Rad). The primer pairs were intron-spanning to avoid amplification of genomic DNA.Generation of Anti-HCN3 Antibody and Western Blot—Polyclonal rabbit antibody directed against the C-terminal (amino acids 552-779) region of murine HCN3 was generated by immunization (Gramsch Laboratories; Schwabhausen, Germany) with a His-Tag fusion protein expressed and purified using the QiaExpress-Kit (Qiagen, Germany) and affinity-purified using the Amino-Link-Kit (Pierce).To determine the specificity of the anti-HCN3 antibody, membrane proteins were isolated from HEK293 cells transfected with mHCN1, mHCN2, mHCN3, or hHCN4 as described previously (22Much 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 (145) Google Scholar) and subjected to Western blot analysis with anti-HCN3. HEK293T cells transduced with LV-HCN3 were analyzed using the same protocol.Tissue from various mouse brain regions was dissected and snap frozen in liquid nitrogen. Samples were homogenized on dry ice using a mortar and pestle, boiled in lysis buffer (2% SDS, 50 mm Tris) for 10 min, and centrifuged (15 min at 16,000 × g) to remove cell debris. Proteins were separated using 7% SDS-PAGE followed by Western blot analysis according to standard procedures. The antibodies anti-HCN3 (1:2,000) and anti-tubulin (1:400; Dianova, Germany) were used.For deglycosylation, homogenates or membrane proteins were first denatured in the presence of 0.5% SDS/0,1 M 2-mercaptoethanol for 3 min at 95 °C and then incubated in 50 mm PBS/1% Nonidet P-40 and 500U of peptide/N-glycosidase F (Roche) for 1 h at 37 °C. Subsequently, proteins were subjected to Western blot analysis.Electrophysiology—Currents were measured 2-3 days after infection with recombinant lentivirus using whole-cell patch clamp technique. Patches were equilibrated for at least 2 min before starting experiments to minimize run down of current. The standard extracellular solution was composed of 110 mm NaCl, 0.5 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, 30 mm KCl, pH = 7.4, adjusted with NaOH. When required, ivabradine (S 16257-2) and Cs+ were added to the extracellular solution by dissolving a stock 10 mm solution to the final 30 μm and a stock 1 m to the final 2 mm, respectively. The intracellular solution contained 130 mm KCl, 10 mm NaCl, 3 mm MgATP, 0.5 mm MgCl2, 1 mm EGTA, 5 mm HEPES, pH = 7.4, adjusted with KOH. For determining the cAMP and cGMP sensitivity of HCN channels intracellular solution was supplemented with 0.5 mm cAMP or cGMP. The extracellular solutions were exchanged by a local solution exchanger. The different solutions reached the cell membrane within less than 100 ms. All recordings were obtained at room temperature. 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) and Origin (Origin Lab Corporation). For determination of the voltage of half-maximal activation (V0.5) currents were elicited by hyperpolarizing the membrane for 3 s to voltages ranging from -140 to -20 mV (in 10-mV increments) from a holding potential of -40 mV followed by a 500 ms step to -140 mV. Amplitude of tail currents, determined immediately after the disappearance of the capacitance transient, were normalized to the maximal current at -140 mV (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/[1 - exp([Vm - V0.5]/k)], where Imin is an offset caused by a non-zero holding current, Vm is the test potential, V0.5 is the voltage of half-maximal activation, and k is the slope factor. Time constants of channel activation (τact) of HCN2 and HCN3 channels were determined by monoexponential function fitting the current evoked during hyperpolarizing voltage pulses to appropriate voltage. As has been described earlier the initial lag in the activation of HCN channel currents was excluded from the fitting procedure (23Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (312) Google Scholar). For determination of contribution of instantaneous current (Iinst) to the total Ih current, the size of instantaneous as well as steady-state current elicited by a 3-s pulse to -140 mV was determined and corrected for the leak, and the amplitude of instantaneous current was normalized with respect to the amplitude of the steady-state current.Statistics—All values are given as mean ± S.E., and n is the number of experiments. An unpaired Student's t test was performed for the comparison between two groups. Values of p < 0.05 were considered significant.RESULTSExpression of HCN3 in Mouse Heart and Brain—RT-PCR revealed that HCN3 transcripts are present in the mouse brain and heart ventricle (Fig. 1A). To study the expression levels of the channel in distinct regions of the brain we raised a polyclonal antibody against the unique C terminus of the protein. The antibody recognized a 86-kDa protein (corresponding to the theoretical Mw of mHCN3) in membrane fractions of HEK293 cells transfected with mHCN3 (Fig. 1B). This band was not observed in non-transfected cells and in cells transfected with HCN1, HCN2, or HCN4, demonstrating the specificity of the anti-HCN3 antibody (Fig. 1B). In mouse brain the antibody detected a slightly bigger protein (∼90 kDa) than in HEK293 cells transfected with mHCN3 (Fig. 1C). This size difference was because of N-linked glycosylation because it disappeared after incubation with N-glycosidase F (Fig. 1D). The expression levels of HCN3 were high in the olfactory bulb and hypothalamus, intermediate in amygdala and hippocampus, and low in retina and cortex (Fig. 1C). In cerebellum, HCN3 was only faintly, if at all, detectable (Fig. 1C). Similarly, the expression of HCN3 protein in heart tissue was below detection level (not shown).Fig. 1Analysis of HCN3 expression in mouse tissues. A, RT-PCR with HCN3-specific primers detects HCN3 mRNA in total mouse brain (Br) and heart ventricle (Ve); Ø indicates control reactions without cDNA. B, immunoblot with anti-HCN3 performed with membrane protein preparations of untransfected and HCN1-, HCN2-, HCN3-, or HCN4-transfected HEK293 cells (16 μg of protein/lane). C, Western blot of homogenates from mouse retina (Re), olfactory bulb (Ob), amygdala (Am), hippocampus (Hi), cortex (Cx), hypothalamus (Hy), and cerebellum (Ce) (top panel). 55 μg of protein was loaded per lane except for cerebellum (110 μg). In the first lane, membrane fractions of HCN3-expressing HEK293 cells (20 μg) were loaded as a positive control. The same blot probed with anti-tubulin antibody (bottom panel). D, analysis of N-linked glycosylation. Homogenates from mouse olfactory bulb (100 μg) and membrane fractions of HCN3-expressing HEK293 cells (40 μg; HCN3) were incubated for 1 h in the presence (+) or in the absence (Ø) of peptide/N-deglycosylase F (PNG-F). Proteins were then analyzed by immunoblot with anti-HCN3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lentiviral Expression of HCN3 in HEK293T Cells—In previous attempts using chemical transfection methods only a small percentage of cells exhibited Ih, with current densities too low for a detailed biophysical analysis. To circumvent this problem we made use of a state-of-the-art lentiviral expression system (21Pfeifer A. Brandon E.P. Kootstra N. Gage F.H. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11450-11455Crossref PubMed Scopus (187) Google Scholar). The vector design allows the coexpression of the gene of interest together with EGFP (via an IRES sequence) to facilitate the screening of the positive cells. Fig. 2A shows an example of HEK293T cells infected with LV-HCN3. About 30% of the cells revealed green fluorescence. The successful expression of the HCN3 protein was confirmed by Western blot. When a membrane preparation from infected cells was used, a specific band of ∼86 kDa was observed, corresponding to the non-glycosylated HCN3 protein (Fig. 2B).HCN3 Encodes a Hyperpolarization-activated Current with Slow Kinetics—EGFP-positive cells were used for electrophysiological analysis. HCN3 currents were compared in side-by-side experiments with Ih from HEK293T cells infected with a lentiviral HCN2 expression vector. The current density obtained for HCN3 was approximately five times smaller than the current density in HCN2-expressing cells (27 ± 5 pA/pF, n = 29 for HCN3 versus 139 ± 26 pA/pF, n = 26 for HCN2). Fig. 3, A and B show representative whole-cell current traces elicited by a family of hyperpolarizing steps from a holding potential of -40 mV. Both currents are composed of a fast Iinst and a slowly activating sigmoidal component. The relative amplitude of the Iinst was consistently larger in HCN3 (13 ± 2% of total current, n = 19) than in HCN2 (5.0 ± 0.7%, n = 20). HCN3 activated and deactivated with significantly slower kinetics than HCN2. The activation time constants at a fully activating membrane potential (-140 mV) were τact = 470 ± 30 ms (n = 13) for HCN3 and τact = 330 ± 30 ms (n = 9) for HCN2. By contrast, HCN2 and HCN3 did not differ from each other, with respect to their voltage of half-maximal activation (V0.5 = -95 mV, Fig. 3D). In addition, the reversal potential obtained from the I-V curve of the fully activated channel was not different between the two channels (-27 mV, data not shown) indicating that both channels share the same ion selectivity.Fig. 3Current-voltage (I-V) characteristics of HCN3 and HCN2 overexpressed in HEK293T cells using lentiviral vectors. A and B, family of current traces of HCN3 (A) and HCN2 (B) elicited by stepping from a holding potential of -40 mV to hyperpolarizing voltages from -140 to -20 mV for 3 s; followed by a 500-ms step to -140 mV. C, voltage dependence of activation time constant τact for HCN3 (•) and HCN2 (○). D, I-V characteristics of HCN3 (•) and HCN2 (○) as derived from the tail current analysis of currents like those in A and B, respectively. Tail currents immediately after the disappearance of the capacitance transient (arrow) were normalized to maximal current at -140 mV (Imax) and plotted as a function of the preceding membrane potential. Solid lines are fits to the Boltzmann function. The fitting parameters were obtained as follows: HCN3, V0.5 = -95 ± 1 mV; k = 9.6 ± 0.6 mV (n = 18); HCN2, V0.5 = -95 ± 1 mV; k = 7 ± 1 mV (n = 13).View Large Image Figure ViewerDownload Hi-res image Download (PPT)HCN3 currents revealed the typical pharmacological profile of native and heterologously expressed Ih channels. The channel was readily blocked by 2 mm extracellular Cs+ (Fig. 4A). Likewise, the bradycardic drug ivabradine (6Bois P. Bescond J. Renaudon B. Lenfant J. Br. J. Pharmacol. 1996; 118: 1051-1057Crossref PubMed Scopus (263) Google Scholar) almost completely inhibited the fully activated current at a concentration of 30 μm. This concentration was used previously to block specifically Ih in sino-atrial node cells (24Bucchi A. Baruscotti M. DiFrancesco D. J. Gen. Physiol. 2002; 120: 1-13Crossref PubMed Scopus (235) Google Scholar) (Fig. 4B).Fig. 4Extracellular Cs+ and ivabradine block HCN3 currents. A, current traces at -140 mV recorded in the presence and absence of 2 mm extracellular Cs+. B, blocking effect of 30 μm ivabradine on the HCN3 current at -140 mV (n = 5 and 6, respectively).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cyclic Nucleotides Shift the Activation Curve of HCN3 to More Negative Voltages—We next went on to test the effect of cAMP on lentivirally expressed HCN channels. Fig. 5, A and B show representative normalized current traces of fully activated HCN2 and HCN3 channels (at -140 mV) obtained either with control pipette solution or after perfusion with 0.5 mm cAMP. In accordance with previous studies using chemical transfection methods (25Biel M. Schneider A. Wahl C. Trends Cardiovasc. Med. 2002; 12: 206-212Crossref PubMed Scopus (203) Google Scholar) cAMP speeded up the activation kinetics of HCN2 (τact (-cAMP) = 330 ± 30 ms (n = 9); τact (+cAMP) = 165 ± 7 ms (n = 12); Fig. 5E). By contrast, perfusion with 0.5 mm cAMP did not significantly alter the kinetics of the HCN3 current (τact (-cAMP) = 470 ± 30 ms (n = 13); τact (+cAMP) = 510 ± 30 ms (n = 11)). Cyclic AMP shifts the voltage dependence of all HCN channels characterized so far to more depolarized potentials. Indeed, the voltage curve of lentivirally expressed HCN2 was about +10 mV more positive in the presence than in the absence of cAMP (V0.5 = -95 ± 1 mV versus -85 ± 1 mV (n = 13 and 9, respectively); Fig. 5F). Interestingly, the effect of cAMP on the HCN3 activation curves (Fig. 5D) was profoundly different from that on HCN2 (Fig. 5C). cAMP reduced the steepness of the I-V curve (k (-cAMP) = 9.6 ± 0.6 mV (n = 18); k (+cAMP) = 11.8 ± 0.7 mV (n = 15)). As a consequence, the half-maximal activation potential was slightly but significantly shifted to more negative values (V0.5 = -95 ± 1 mV versus -100 ± 2 mV (n = 18 and 15, respectively); p < 0.05; Fig. 5F).Fig. 5Modulation of HCN2 and HCN3 current by cAMP. A and B, normalized current traces of HCN2 (A) and HCN3 (B) in the absence and presence of 0.5 mm intracellular cAMP. Currents were evoked by a hyperpolarizing step to -140 mV from a holding potential of -40 mV. The leak current was subtracted. C and D, activation curves of HCN2 (C) and HCN3 (D) in the absence (•) and presence (○) of cAMP. Solid lines represent fit to the Boltzmann function. Parameters in the presence of cAMP are as follows: HCN2, V0.5 = -85 ± 1 mV; k = 8 ± 1 mV (n = 9); HCN3, V0.5 = -100 ± 2 mV; k = 11.8 ± 0.7 mV (n = 15). For fitting parameter in the absence of cAMP see Fig. 3. E, activation constants of the fully activated channels obtained at -140 mV (τact-140). HCN3, τact-140 = 470 ± 30 ms and 510 ± 30 ms in the absence (n = 13) and presence (n = 11) of cAMP, respectively; HCN2, τact-140 = 330 ± 30 ms and 165 ± 7 ms in the absence (n = 9) and presence (n = 12) of cAMP, respectively. F, bar diagram illustrating the opposite shift of V0.5 induced by cAMP in HCN3 and HCN2, respectively. *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cyclic GMP modulated HCN3 currents in a similar manner as cAMP did. Intracellular application of 0.5 mm cGMP did not significantly alter the kinetics of the fully activated HCN3 current (τact (-cGMP) = 470 ± 30 ms (n = 13); τact (+cGMP) = 520 ± 50 ms (n = 17) at -140 mV; Fig. 6A). At less hyperpolarizing conditions cGMP tended to slow down HCN3 current kinetics (Fig. 6B). However, this effect was not statistically significant (p > 0.05). In the presence of cAMP the same behavior was observed (data not shown). Like cAMP, cGMP shifted the voltage dependence of HCN3 activation slightly to more negative values (V0.5 = -95 ± 1 mV versus -101 ± 1 mV (n = 18 and 17 in the absence and presence of cGMP, respectively); Fig. 6C). This effect was statistically significant (p < 0.05) and resulted from the change in the steepness of the I-V curve (k (-cGMP) = 9.6 ± 0.6 mV (n = 18); k (+cGMP) = 11.3 ± 0.6 mV (n = 17)).Fig. 6Modulation of HCN3 current by cGMP. A, normalized current traces of HCN3 in the absence and presence of 0.5 mm intracellular cGMP. Currents were evoked by a hyperpolarizing step to -140 mV from a holding potential of -40 mV. The leak current was subtracted. B, voltage dependence of activation time constant τact for HCN3 in the absence (•) and presence (○) of 0.5 mm cGMP. C, activation curves of HCN3 in the absence (•) and presence (○) of cGMP. Solid lines represent fits to the Boltzmann function. Parameters in the presence of cGMP are as follows: V0.5 = -101 ± 1 mV; k = 11.3 ± 0.6 mV (n = 17). For fitting parameter in the absence of cGMP see Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Thus, with respect to V0.5 values, cyclic nucleotides regulated HCN2 and HCN3 in opposite direction. Because the voltage dependence of HCN2 activation determined in our study was entirely consistent with previous data on that channel the unique behavior of HCN3 was genuine and not an artifact resulting from the lentiviral expression system used.DISCUSSIONOur knowledge of the HCN channel family has increased dramatically since the first cloning of the channels 7 years ago (20Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scop" @default.
• W2094516452 created "2016-06-24" @default.
• W2094516452 creator A5013075360 @default.
• W2094516452 creator A5034719330 @default.
• W2094516452 creator A5071341634 @default.
• W2094516452 creator A5071705919 @default.
• W2094516452 creator A5087875400 @default.
• W2094516452 creator A5089190614 @default.
• W2094516452 date "2005-07-01" @default.
• W2094516452 modified "2023-10-16" @default.
• W2094516452 title "The Murine HCN3 Gene Encodes a Hyperpolarization-activated Cation Channel with Slow Kinetics and Unique Response to Cyclic Nucleotides" @default.
• W2094516452 cites W1974273055 @default.
• W2094516452 cites W1977529073 @default.
• W2094516452 cites W1983804855 @default.
• W2094516452 cites W1985663331 @default.
• W2094516452 cites W1988174768 @default.
• W2094516452 cites W1989652455 @default.
• W2094516452 cites W1994962594 @default.
• W2094516452 cites W1998093640 @default.
• W2094516452 cites W2002811618 @default.
• W2094516452 cites W2004781289 @default.
• W2094516452 cites W2005264220 @default.
• W2094516452 cites W2010973571 @default.
• W2094516452 cites W2014660839 @default.
• W2094516452 cites W2014880992 @default.
• W2094516452 cites W2023001234 @default.
• W2094516452 cites W2026641055 @default.
• W2094516452 cites W2037699648 @default.
• W2094516452 cites W2043805631 @default.
• W2094516452 cites W2046408227 @default.
• W2094516452 cites W2050218537 @default.
• W2094516452 cites W2054498084 @default.
• W2094516452 cites W2078349199 @default.
• W2094516452 cites W2086190065 @default.
• W2094516452 cites W2091122073 @default.
• W2094516452 cites W2091651584 @default.
• W2094516452 cites W2094531569 @default.
• W2094516452 cites W2102160264 @default.
• W2094516452 cites W2109153798 @default.
• W2094516452 cites W2118621245 @default.
• W2094516452 cites W2120400290 @default.
• W2094516452 cites W2153500492 @default.
• W2094516452 cites W2153822662 @default.
• W2094516452 cites W2154166729 @default.
• W2094516452 cites W2158022157 @default.
• W2094516452 cites W2171502806 @default.
• W2094516452 cites W2171569887 @default.
• W2094516452 cites W2173794755 @default.
• W2094516452 cites W4233293499 @default.
• W2094516452 doi "https://doi.org/10.1074/jbc.m502696200" @default.
• W2094516452 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15923185" @default.
• W2094516452 hasPublicationYear "2005" @default.
• W2094516452 type Work @default.
• W2094516452 sameAs 2094516452 @default.
• W2094516452 citedByCount "103" @default.
• W2094516452 countsByYear W20945164522012 @default.
• W2094516452 countsByYear W20945164522013 @default.
• W2094516452 countsByYear W20945164522014 @default.
• W2094516452 countsByYear W20945164522015 @default.
• W2094516452 countsByYear W20945164522016 @default.
• W2094516452 countsByYear W20945164522017 @default.
• W2094516452 countsByYear W20945164522018 @default.
• W2094516452 countsByYear W20945164522019 @default.
• W2094516452 countsByYear W20945164522021 @default.
• W2094516452 countsByYear W20945164522022 @default.
• W2094516452 countsByYear W20945164522023 @default.
• W2094516452 crossrefType "journal-article" @default.
• W2094516452 hasAuthorship W2094516452A5013075360 @default.
• W2094516452 hasAuthorship W2094516452A5034719330 @default.
• W2094516452 hasAuthorship W2094516452A5071341634 @default.
• W2094516452 hasAuthorship W2094516452A5071705919 @default.
• W2094516452 hasAuthorship W2094516452A5087875400 @default.
• W2094516452 hasAuthorship W2094516452A5089190614 @default.
• W2094516452 hasBestOaLocation W20945164521 @default.
• W2094516452 hasConcept C104317684 @default.
• W2094516452 hasConcept C121332964 @default.
• W2094516452 hasConcept C12554922 @default.
• W2094516452 hasConcept C131453863 @default.
• W2094516452 hasConcept C148898269 @default.
• W2094516452 hasConcept C153911025 @default.
• W2094516452 hasConcept C170493617 @default.
• W2094516452 hasConcept C185592680 @default.
• W2094516452 hasConcept C2778524494 @default.
• W2094516452 hasConcept C2780965148 @default.
• W2094516452 hasConcept C50254741 @default.
• W2094516452 hasConcept C512185932 @default.
• W2094516452 hasConcept C55493867 @default.
• W2094516452 hasConcept C62520636 @default.
• W2094516452 hasConcept C66974803 @default.
• W2094516452 hasConcept C71240020 @default.
• W2094516452 hasConcept C86803240 @default.
• W2094516452 hasConcept C95444343 @default.
• W2094516452 hasConceptScore W2094516452C104317684 @default.
• W2094516452 hasConceptScore W2094516452C121332964 @default.
• W2094516452 hasConceptScore W2094516452C12554922 @default.
• W2094516452 hasConceptScore W2094516452C131453863 @default.
• W2094516452 hasConceptScore W2094516452C148898269 @default.
• W2094516452 hasConceptScore W2094516452C153911025 @default.

• next