Title: Absence of Direct Cyclic Nucleotide Modulation of mEAG1 and hERG1 Channels Revealed with Fluorescence and Electrophysiological Methods
Abstract: Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (Kd ≥ 51 μm) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K+ channels might harbor a binding site for a ligand yet to be uncovered. Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (Kd ≥ 51 μm) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K+ channels might harbor a binding site for a ligand yet to be uncovered. The EAG family of K+ channels comprises ether-à-go-go (EAG), 2The abbreviations used are: EAGether-à-go-goERGEAG-related geneCNBDcyclic nucleotide binding domainELKEAG-like K+ channelHCNhyperpolarization-activated cyclic nucleotide-modulatedCNGcyclic nucleotide-gatedMBPmaltose-binding protein. EAG-related gene (ERG), and EAG-like (ELK) K+ channel subfamilies (1Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar) with diverse tissue expression patterns and physiological functions (reviewed in Ref. 2Bauer C.K. Schwarz J.R. J. Membr. Biol. 2001; 182: 1-15Crossref PubMed Scopus (105) Google Scholar). mEAG channels are overexpressed in tumor tissues (3Farias L.M. Ocaña D.B. Diaz L. Larrea F. Avila-Chávez E. Cadena A. Hinojosa L.M. Lara G. Villanueva L.A. Vargas C. Hernández-Gallegos E. Camacho-Arroyo I. Dueñas-González A. Pérez-Cárdenas E. Pardo L.A. Morales A. Taja-Chayeb L. Escamilla J. Sánchez-Peña C. Camacho J. Cancer Res. 2004; 64: 6996-7001Crossref PubMed Scopus (121) Google Scholar, 4Hemmerlein B. Weseloh R.M. Mello de Queiroz F. Knötgen H. Sánchez A. Rubio M.E. Martin S. Schliephacke T. Jenke M. Heinz-Joachim-Radzun Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 41Crossref PubMed Scopus (208) Google Scholar), where they are involved in regulation of tumor progression (5Ouadid-Ahidouch H. Le Bourhis X. Roudbaraki M. Toillon R.A. Delcourt P. Prevarskaya N. Receptors Channels. 2001; 7: 345-356PubMed Google Scholar, 6Pardo L.A. del Camino D. Sánchez A. Alves F. Brüggemann A. Beckh S. Stühmer W. EMBO J. 1999; 18: 5540-5547Crossref PubMed Scopus (361) Google Scholar). Inhibition of the EAG channel expression by RNAi interference (7Weber C. Mello de Queiroz F. Downie B.R. Suckow A. Stühmer W. Pardo L.A. J. Biol. Chem. 2006; 281: 13030-13037Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), application of channel blockers (8Mello de Queiroz F. Suarez-Kurtz G. Stühmer W. Pardo L.A. Mol. Cancer. 2006; 5: 42Crossref PubMed Scopus (87) Google Scholar, 9Gavrilova-Ruch O. Schönherr K. Gessner G. Schönherr R. Klapperstück T. Wohlrab W. Heinemann S.H. J. Membr. Biol. 2002; 188: 137-149Crossref PubMed Scopus (109) Google Scholar), and monoclonal antibody that selectively inhibits currents from EAG channels (10Gómez-Varela D. Zwick-Wallasch E. Knötgen H. Sánchez A. Hettmann T. Ossipov D. Weseloh R. Contreras-Jurado C. Rothe M. Stühmer W. Pardo L.A. Cancer Res. 2007; 67: 7343-7349Crossref PubMed Scopus (172) Google Scholar) decreased cell proliferation in tumor tissues. ether-à-go-go EAG-related gene cyclic nucleotide binding domain EAG-like K+ channel hyperpolarization-activated cyclic nucleotide-modulated cyclic nucleotide-gated maltose-binding protein. ERG channels are best known for their function in the heart. Because of their unique physiological properties, fast inactivation, and slow deactivation, ERG channels are major contributors to the repolarization phase of the cardiac action potential (11Spector P.S. Curran M.E. Zou A. Keating M.T. Sanguinetti M.C. J. Gen. Physiol. 1996; 107: 611-619Crossref PubMed Scopus (377) Google Scholar, 12Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (668) Google Scholar, 13Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1099) Google Scholar, 14Schönherr R. Heinemann S.H. J. Physiol. 1996; 493: 635-642Crossref PubMed Scopus (263) Google Scholar). Mutations in the ERG channels and administration of ERG channel blockers, such as class III antiarrhythmic drugs, cause long QT syndrome, a potentially lethal cardiac arrhythmia characterized by a prolonged cardiac action potential (15Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (1996) Google Scholar, 16Kiehn J. Lacerda A.E. Wible B. Brown A.M. Circulation. 1996; 94: 2572-2579Crossref PubMed Scopus (187) Google Scholar, 17Sanguinetti M.C. Jurkiewicz N.K. Scott A. Siegl P.K. Circ. Res. 1991; 68: 77-84Crossref PubMed Scopus (283) Google Scholar, 18Li X. Xu J. Li M. J. Biol. Chem. 1997; 272: 705-708Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 19Zhou Z. Gong Q. Epstein M.L. January C.T. J. Biol. Chem. 1998; 273: 21061-21066Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). In addition to their role in cardiac excitability, ERG channels also regulate proliferation of tumor cells (20Smith G.A. Tsui H.W. Newell E.W. Jiang X. Zhu X.P. Tsui F.W. Schlichter L.C. J. Biol. Chem. 2002; 277: 18528-18534Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 21Meyer R. Heinemann S.H. J. Physiol. 1998; 508: 49-56Crossref PubMed Scopus (105) Google Scholar, 22Crociani O. Guasti L. Balzi M. Becchetti A. Wanke E. Olivotto M. Wymore R.S. Arcangeli A. J. Biol. Chem. 2003; 278: 2947-2955Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The physiological role of ELK channels is not well understood, however, early reports suggest their possible involvement in the regulation of neuronal excitability (23Becchetti A. De Fusco M. Crociani O. Cherubini A. Restano-Cassulini R. Lecchi M. Masi A. Arcangeli A. Casari G. Wanke E. Eur. J. Neurosci. 2002; 16: 415-428Crossref PubMed Scopus (36) Google Scholar). K+ channels in the EAG family are structurally related to the cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-modulated (HCN) K+ channels (1Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar, 24Guy H.R. Durell S.R. Warmke J. Drysdale R. Ganetzky B. Science. 1991; 254: 730Crossref PubMed Scopus (98) Google Scholar). All of these channels contain a CNBD in their C-terminal region. Unlike HCN and CNG channels whose regulation by direct binding of cyclic nucleotides to the CNBD is well established (25DiFrancesco D. Tortora P. Nature. 1991; 351: 145-147Crossref PubMed Scopus (649) Google Scholar, 26Fesenko E.E. Kolesnikov S.S. Lyubarsky A.L. Nature. 1985; 313: 310-313Crossref PubMed Scopus (814) Google Scholar, 27Haynes L. Yau K.W. Nature. 1985; 317: 61-64Crossref PubMed Scopus (155) Google Scholar, 28Ludwig A. Zong X. Jeglitsch M. Hofmann F. Biel M. Nature. 1998; 393: 587-591Crossref PubMed Scopus (787) Google Scholar, 29Nakamura T. Gold G.H. Nature. 1987; 325: 442-444Crossref PubMed Scopus (849) Google Scholar, 30Finn J.T. Grunwald M.E. Yau K.W. Annu. Rev. Physiol. 1996; 58: 395-426Crossref PubMed Scopus (281) Google Scholar, 31Zagotta W.N. Olivier N.B. Black K.D. Young E.C. Olson R. Gouaux E. Nature. 2003; 425: 200-205Crossref PubMed Scopus (486) Google Scholar, 32Santoro 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 (584) Google Scholar), regulation of the EAG family of K+ channels by the direct binding of cyclic nucleotides is controversial. It has been reported that EAG channels in mouse (33Robertson G.A. Warmke J.M. Ganetzky B. Neuropharmacology. 1996; 35: 841-850Crossref PubMed Scopus (95) Google Scholar), rat (34Ludwig J. Terlau H. Wunder F. Brüggemann A. Pardo L.A. Marquardt A. Stühmer W. Pongs O. EMBO J. 1994; 13: 4451-4458Crossref PubMed Scopus (153) Google Scholar), and bovine retina (35Frings S. Brüll N. Dzeja C. Angele A. Hagen V. Kaupp U.B. Baumann A. J. Gen. Physiol. 1998; 111: 583-599Crossref PubMed Scopus (74) Google Scholar) and ERG channels in humans (36Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2152) Google Scholar) are not regulated by cyclic nucleotides. However, in similar studies other groups have shown that EAG channels in Drosophila (37Brüggemann A. Pardo L.A. Stühmer W. Pongs O. Nature. 1993; 365: 445-448Crossref PubMed Scopus (198) Google Scholar, 38Zhong Y. Wu C.F. J. Neurosci. 1993; 13: 4669-4679Crossref PubMed Google Scholar) and ERG channels in humans (39Cui J. Kagan A. Qin D. Mathew J. Melman Y.F. McDonald T.V. J. Biol. Chem. 2001; 276: 17244-17251Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 40Cui J. Melman Y. Palma E. Fishman G.I. McDonald T.V. Curr. Biol. 2000; 10: 671-674Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) are regulated by cAMP. Most of the above mentioned studies were performed in a whole-cell or two-electrode voltage clamp configuration. In either of these configurations it is difficult if not impossible to control the concentration of the applied cyclic nucleotides and differentiate between direct effect of cyclic nucleotides on the EAG and ERG channels and secondary effects through signaling pathways regulated by cyclic nucleotides. To resolve this controversy we took a direct approach by applying cyclic nucleotides directly to the isolated CNBD and membrane patches expressing channels in the inside-out configuration. The direct binding of cAMP and cGMP to the isolated CNBD of the mEAG1 (also known as KCNH1 and Kv10.1) and hERG1 (also known as KCNH2 and Kv11.1) channels was examined with fluorescence-based methods. To demonstrate the validity of our approach, the fluorescence methods were also applied to the isolated CNBD of mHCN2 channels. The effect of cAMP and cGMP on full-length channels was examined by direct application of cyclic nucleotides to inside-out patches expressing mEAG1 and hERG1 channels. The fluorescent-based experiments indicated no binding of the cyclic nucleotides to the CNBD of mEAG1 and only low affinity binding (Kd ≥ 51 μm) of cAMP to the CNBD of hERG1 channels. Direct application of cAMP and cGMP had no effect on the currents recorded from mEAG1 and hERG1 channels. Our results indicate that cAMP and cGMP do not regulate mEAG1 and hERG1 channels by direct binding to the CNBD. To express portions of the C-terminal regions containing the CNBD, the DNA fragments encoding residues 505–702 of the mEAG1 channel (mEAG1-(505–702)), 666–872 of the hERG1 channel (hERG1-(666–872)), and 443–645 of the wild-type and mutant mHCN2 channels with Trp substituted for Leu at the position 586 (mHCN2J and mHCN2J-L586W, respectively) were subcloned into a pETGQ vector (41Chen G.Q. Gouaux E. Protein Eng. 1997; 10: 1061-1066Crossref PubMed Scopus (15) Google Scholar). The constructs were grown in BL21 (DE3) cells at 37 °C. At OD 0.6–0.8, the cell cultures were cooled on ice and induced with 1 m isopropyl-1-thio-β-d-galactopyranoside. After growing overnight at 18 °C, the cells were harvested by centrifugation at 5000 × rpm for 15 min at 4 °C, and the cell pellets were frozen at −80 °C. The cells were then resuspended in a lysis buffer (150 mm KCl, 10% glycerol, 1 mm TCEP, 30 mm HEPES, 1 mm phenylmethylsulfonyl fluoride, and 2.5 mg/ml DNase; pH 7.5) and lysed in an Emulsiflex-C5 (Avestin). Insoluble protein was separated by centrifugation for 45 min at 40,000 × rpm at 4 °C. The protein of interest was then purified from the supernatant by Ni2+-nitrilotriacetic acid chromatography and eluted on a linear gradient to 500 mm imidazole. The 6× His tag was cleaved with thrombin protease (Calbiochem) and separated with size exclusion chromatography. The protein was purified on a Superdex 200 column (Amersham Biosciences) equilibrated with the buffer used for the subsequent experiments (150 mm KCl, 10% glycerol, 1 mm TCEP, 30 mm HEPES; pH 7.5). The purified protein was stored at −80 °C in small aliquots and thawed immediately before the experiments. The molecular weight of the constructs used was verified on Coomassie Blue-stained gels and with mass spectrometry. The protein concentration was determined by absorbance at 280 nm and with RC DC protein assay (Bio-Rad) based on the Lowry method (42Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Both methods gave similar values for the protein concentration. hERG1-(666–872) was eluted at the void volume of the column when expressed in pETGQ vector. In an attempt to increase monodispersity hERG1-(666–872) was subcloned into pHMalc2T vector, a modified version of pMalc2T (New England Biolabs), with maltose-binding protein (MBP) as a tag. The purification steps were carried out as described above except the protein was purified on amylose affinity column (New England Biolabs), instead of the Ni2+ affinity column, and eluted on a linear gradient to 50 mm maltose. hERG1-(666–872) MBP fusion protein (hERG1-(666–872)/MBP) was monodisperse and used for the fluorescence-based assays. Fluorescence intensity was recorded with a Fluorolog 3 spectrophotometer (HORIBA Jobin Yvon) using FluorEssence software. For the experiments with cAMP and cGMP, the sample was excited at 295 nm, and the emission spectrum was recorded from 300 to 500 nm. To account for the decrease in the excitation and emission intensities due to the absorbance, observed fluorescence intensities of the sample and buffer were corrected for the inner filter effect according to Equation 1 (43Lakowicz J.R. Principles Of Fluorescence Spectroscopy. 3rd Ed. Springer, New York, NY2006: 56Google Scholar), Fci=Foi(10(0.1)*A295+0.5*Ai)(Eq. 1) where Fci and Foi are the corrected and observed fluorescence intensities at the i nm wavelength, A295 and Ai are the absorbance recorded at 295 nm and i nm wavelength, respectively. To calculate the final fluorescence intensity, the corrected buffer intensity was subtracted from the corrected sample intensity. Each of the experiments was repeated at least three times. The error bars on the figures correspond to the S.E. For experiments with 8-NBD-cAMP a fluorescent analog of cAMP (BIOLOG, Bremen, Germany), the sample was excited at 470 nm, and the emission spectra were recorded from 480 to 650 nm. The inner filter correction was carried out similar to the experiments with cAMP and cGMP, except the A at 470 nm was used instead of the A at 295 nm in Equation 1. To estimate the binding affinity, plots of the change of the peak fluorescence intensities versus total cyclic nucleotide concentration were analyzed as in Cukkemane et al. (60Cukkemane A. Grüter B. Novak K. Gensch T. Bönigk W. Gerharz T. Kaupp U.B. Seifert R. EMBO Rep. 2007; 8: 749-755Crossref PubMed Scopus (45) Google Scholar). Briefly, binding of a ligand to a receptor was treated as a simple first order reaction, R+L⇀↽RL(Eq. 2) Kd=R×LRL=(Rt-RL)×(Lt-RL)RL(Eq. 3) RL=12(Rt+Lt+Kd)-14(-Rt-Lt-Kd)2-Rt×Rt×Lt(Eq. 4) ΔF=RL×x(Eq. 5) where R, L, and RL are concentrations of the free receptor and ligand, and receptor-ligand complex, respectively; Rt and Lt are total receptor and ligand concentrations; ΔF is the peak fluorescence change, and x is a scaling factor. The data analysis and fitting of the plots was performed in Origin (Microcal Software, Inc). Circular dichroism (CD) spectra were recorded with an Aviv 62A DS spectrometer (Aviv Associates, Lakewood, NJ) at 22 °C using a 1-mm pathlength cuvette. Three scans were averaged for each sample with data acquired every 1 nm. For the CD experiments, the protein was purified in 150 mm potassium phosphate buffer with 10% glycerol, 1 mm TCEP, pH 7.5. The protein concentration used was 20 μm. The cDNA encoding mHCN2 channels in pGEM vector, and hERG1, hERG1-S631A, and mEAG1 channels in pGH19 vector were kindly provided by S. Siegelbaum (Columbia University, New York, NY) and G. Robertson (University of Wisconsin-Madison, Madison, WI), respectively. The cRNA was transcribed using the T7 mMessage mMachine kit (Ambion). Xenopus laevis oocytes were defolliculated and injected with the cRNA as previously described (44Zagotta W.N. Hoshi T. Aldrich R.W. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 7243-7247Crossref PubMed Scopus (122) Google Scholar). Following manual removal of the vitelline membrane, currents were recorded in the inside-out patch configuration (45Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15149) Google Scholar) with an EPC-10 patch-clamp amplifier (HEKA Electronik). Data were acquired with Pulse software (HEKA Elektronik) and analyzed with Igor (WaveMetrics, Inc). Patch pipettes were pulled from borosilicate glass and had resistances of 0.40–1 mΩ after fire polishing. The intracellular (bath) and extracellular (pipette) solutions contained 130 mm KCl, 10 mm HEPES, 0.2 mm EDTA, pH 7.2. cAMP or cGMP were added to the bath solution as indicated. The bath solution was changed with RSC- 100 solution changer (BioLogic). mHCN2 currents were elicited by applying a series of 5-s voltage pulses (ranging from −140 to −70 mV in 10-mV increments) from a holding potential of 0 mV, followed by a 1-s voltage tail pulse to −40 mV. hERG1 currents were elicited by applying a series of 0.25-s voltage pulses (ranging from −100 to +100 mV in 20-mV increments) from a holding potential of −80 mV, followed by a 0.5-s voltage pulse to −100 mV. mEAG1 and hERG1-S631A currents were elicited by applying a series of 0.1-s voltage pulses (ranging from −140 to +50 mV in 10-mV increments) from a holding potential of −100 mV, followed by a 0.5-s voltage pulse to −120 mV. Currents were not leak-subtracted. To determine if the CNBD of the EAG channels contain residues implicated in cyclic nucleotide binding we aligned amino acid sequences of the CNBD of several members of the EAG family of K+ channels with the sequences of proteins that are regulated by direct binding of cyclic nucleotides (Fig. 1). A canonical CNBD contains three α-helices and eight β-strands forming an antiparallel β-roll. The general architecture of the CNBD emerged from the crystal structures of CAP (46Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 2843-2847Crossref PubMed Scopus (159) Google Scholar), PKA (47Su Y. Dostmann W.R. Herberg F.W. Durick K. Xuong N.H. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (345) Google Scholar), and the C terminus of the HCN2 (31Zagotta W.N. Olivier N.B. Black K.D. Young E.C. Olson R. Gouaux E. Nature. 2003; 425: 200-205Crossref PubMed Scopus (486) Google Scholar) and MlotiK1 (48Clayton G.M. Silverman W.R. Heginbotham L. Morais-Cabral J.H. Cell. 2004; 119: 615-627Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) channels. Extensive biochemical studies and the crystal structures of the CNBD identified six invariant residues, indicated by black arrows in Fig. 1. The Gly residues are essential for the structural integrity of the β-roll, Glu and Arg residues take part in the binding of cyclic nucleotides, and the role of Ala is not clear (49Rehmann H. Wittinghofer A. Bos J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 63-73Crossref PubMed Scopus (174) Google Scholar, 50Shabb J.B. Corbin J.D. J. Biol. Chem. 1992; 267: 5723-5726Abstract Full Text PDF PubMed Google Scholar). The three invariant Gly residues are conserved in the EAG family of K+ channels, indicating that the overall fold of the CNBD of the EAG channels is similar to the canonical CNBD structure. The invariant Glu residue is conserved in the hERG1 and is replaced by Asp and Cys in the mEAG1 and mELK2 channels, respectively. In a canonical CNBD, the Glu residue directly binds to the ribose of cyclic nucleotides. Mutations of the Glu residue to Ala in HCN2 channels (51Zhou L. Siegelbaum S.A. Structure. 2007; 15: 655-670Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), to seven different amino acids in CAP (52Belduz A.O. Lee E.J. Harman J.G. Nucleic Acids Res. 1993; 21: 1827-1835Crossref PubMed Scopus (34) Google Scholar, 53Moore J. Kantorow M. Vanderzwaag D. McKenney K. J. Bacteriol. 1992; 174: 8030-8035Crossref PubMed Google Scholar) and to Lys in PKA (54Ogreid D. Døskeland S.O. Gorman K.B. Steinberg R.A. J. Biol. Chem. 1988; 263: 17397-17404Abstract Full Text PDF PubMed Google Scholar) drastically decreased the apparent binding affinity of cyclic nucleotides. Interestingly, substituting Asp for the Glu residue also impaired cAMP binding in CAP (53Moore J. Kantorow M. Vanderzwaag D. McKenney K. J. Bacteriol. 1992; 174: 8030-8035Crossref PubMed Google Scholar). The invariant Arg residue is absent in the EAG family of K+ channels. In other cyclic nucleotide-binding proteins, it forms a salt bridge with the negatively charged phosphate of cyclic nucleotides. Mutations of the Arg residue to Lys or Trp in PKA (54Ogreid D. Døskeland S.O. Gorman K.B. Steinberg R.A. J. Biol. Chem. 1988; 263: 17397-17404Abstract Full Text PDF PubMed Google Scholar, 55Bubis J. Neitzel J.J. Saraswat L.D. Taylor S.S. J. Biol. Chem. 1988; 263: 9668-9673Abstract Full Text PDF PubMed Google Scholar), to several different residues, including Ala, in CAP (52Belduz A.O. Lee E.J. Harman J.G. Nucleic Acids Res. 1993; 21: 1827-1835Crossref PubMed Scopus (34) Google Scholar, 53Moore J. Kantorow M. Vanderzwaag D. McKenney K. J. Bacteriol. 1992; 174: 8030-8035Crossref PubMed Google Scholar, 56Gronenborn A.M. Sandulache R. Gärtner S. Clore G.M. Biochem. J. 1988; 253: 801-807Crossref PubMed Scopus (19) Google Scholar), and to Gln in MlotiK1 channels (57Nimigean C.M. Pagel M.D. J. Mol. Biol. 2007; 371: 1325-1337Crossref PubMed Scopus (19) Google Scholar) abolished cAMP binding. Mutation of the Arg residue to Ala in MlotiK1 and HCN2 channels, and to neutral and negatively charged residues in CNGA1 channels drastically decreased apparent cyclic nucleotide binding affinity (51Zhou L. Siegelbaum S.A. Structure. 2007; 15: 655-670Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 58Altieri S.L. Clayton G.M. Silverman W.R. Olivares A.O. De la Cruz E.M. Thomas L.R. Morais-Cabral J.H. J. Mol. Biol. 2008; 381: 655-669Crossref PubMed Scopus (28) Google Scholar, 59Tibbs G.R. Liu D.T. Leypold B.G. Siegelbaum S.A. J. Biol. Chem. 1998; 273: 4497-4505Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The invariant Glu and Arg residues are essential for the binding of cyclic nucleotides; however, there are exceptions. Even though Epac1, a cAMP-dependent small G-protein Rap activator, is known to bind cAMP with high affinity, it has Gln instead of the invariant Glu residue (49Rehmann H. Wittinghofer A. Bos J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 63-73Crossref PubMed Scopus (174) Google Scholar). Mutating the invariant Arg to Ala in MlotiK1 and HCN2 channels significantly decreases cyclic nucleotide binding affinity, yet, still allows for the cyclic nucleotide modulation of the channels (51Zhou L. Siegelbaum S.A. Structure. 2007; 15: 655-670Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 58Altieri S.L. Clayton G.M. Silverman W.R. Olivares A.O. De la Cruz E.M. Thomas L.R. Morais-Cabral J.H. J. Mol. Biol. 2008; 381: 655-669Crossref PubMed Scopus (28) Google Scholar). Thus, the absence of the invariant Glu or Arg residues would be expected to decrease binding affinity of cyclic nucleotides; however, it will not necessarily prevent binding. To investigate binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels, mEAG1-(505–702) and hERG1-(666–872) MBP fusion constructs were grown in BL21 (DE3) cells and purified with affinity and size-exclusion chromatography. As a positive control for cyclic nucleotide binding in our assays we used mHCN2J-L586W with a Trp residue substituted for the Leu located on the P-helix near the cyclic nucleotide binding pocket. All of the constructs were monodisperse on size exclusion chromatographs, indicating that the protein was not aggregated and likely properly folded (Fig. 2A). The exact molecular weight of the proteins was confirmed with mass spectroscopy. In addition to the CNBD, mEAG1-(505–702), hERG1-(666–872), and mHCN2J-L586W contain the A′-F′ α-helices, spanning the C-linker region between the end of the last transmembrane domain and the CNBD. To further assess the folding of the CNBD, we measured CD spectra for mHCN2J-L586W and mEAG1-(505–702) proteins. The CD spectra indicated a similar structured fold for mHCN2J-L586W and mEAG1-(505–702) (Fig. 2B). Addition of 100 μm cAMP to mHCN2J-L586W increased α-helical content (Fig. 2C) and had no effect on the CD spectrum of mEAG1-(505–702) (Fig. 2D). This suggests that binding of cAMP causes a conformational rearrangement of the CNBD in mHCN2 channels and has little effect on the structure of the CNBD in mEAG1 channels. Increase in the α-helical content upon binding of cAMP was also reported for the isolated CNBD of MlotiK1 channels (60Cukkemane A. Grüter B. Novak K. Gensch T. Bönigk W. Gerharz T. Kaupp U.B. Seifert R. EMBO Rep. 2007; 8: 749-755Crossref PubMed Scopus (45) Google Scholar). Fluorescence of Trp residues has been widely used as an indicator of macromolecular interactions and ligand binding due to its environmental sensitivity (43Lakowicz J.R. Principles Of Fluorescence Spectroscopy. 3rd Ed. Springer, New York, NY2006: 56Google Scholar, 60Cukkemane A. Grüter B. Novak K. Gensch T. Bönigk W. Gerharz T. Kaupp U.B. Seifert R. EMBO Rep. 2007; 8: 749-755Crossref PubMed Scopus (45) Google Scholar). To determine if cyclic nucleotides bind to the isolated CNBD of the mEAG1 channels we took advantage of the endogenous Trp residue at position 649 that, based on a homology model of mEAG1 channels, is located in the P helix neighboring the putative cyclic nucleotide binding pocket (Fig. 3A). Binding of cyclic nucleotides in the vicinity of the Trp residue would be expected to change the Trp fluorescence. We first explored if Trp fluorescence can report on cyclic nucleotide binding to the CNBD of mHCN2 channels that are known to directly bind cyclic nucleotides. There are no endogenous Trp residues in the CNBD of the mHCN2 channels. Therefore, we substituted Trp for Leu residue at the position 586, analogous to 649 in mEAG1 channels, on the P-helix (mHCN2J-L586W). While wild-type mHCN2J showed very little fluorescence upon excitation at 295 nm (supplemental Fig. S1A), mHCN2J-L586W displayed a robust fluorescent signal with a peak at 342 nm (Fig. 3B). The fluorescence intensity decreased with increasing concentration of applied cAMP (Fig. 3, B and C) and cGMP (Fig. 3C). The decrease in the fluorescence intensity is specific to cyclic nucleotide binding to the CNBD as application of cAMP to free tryptophan in solution showed no dose-dependent quenching (supplemental Fig. 1B). To determine cyclic nucleotide binding affinity, we plotted the change in the peak fluorescence intensity versus the total cyclic nucleotide concentration (Fig. 3C). The dose response curves were fitted with Equation 4 as described under "Experimental Procedures." The analysis revealed the binding affinities of 13 ± 2 μm for cAMP and 62 ± 23 μm for cGMP. These affinities are an order of magnitude lower than the cyclic nucleotide binding affinities of the isolated CNBD of MlotiK1 (60Cukkemane A.