Portal de Periódicos da CAPES

Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

2009; Elsevier BV; Volume: 284; Issue: 26 Linguagem: Inglês

10.1074/jbc.m109.001842

1083-351X

Regina Goetz, Katarzyna Dover, Fernanda Laezza, Nataly Shtraizent, Xiao Huang, Dafna Tchetchik, Anna V. Eliseenkova, Chong Xu, Thomas A. Neubert, David M. Ornitz, Mitchell Goldfarb, Moosa Mohammadi,

Wnt/β-catenin signaling in development and cancer

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs. Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs. Voltage-gated sodium channels (Nav) 3The abbreviations used are: Navvoltage-gated sodium channelFGFfibroblast growth factorFGFRFGF receptorFHFFGF homologous factorAISaxon initial segmentSPRsurface plasmon resonanceGFPgreen fluorescent protein. 3The abbreviations used are: Navvoltage-gated sodium channelFGFfibroblast growth factorFGFRFGF receptorFHFFGF homologous factorAISaxon initial segmentSPRsurface plasmon resonanceGFPgreen fluorescent protein. produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. These channels are heteromeric membrane proteins composed of an α subunit, which is sufficient for channel gating, and one or more auxiliary β subunits, which tune voltage dependence and kinetics of channel gating (for review, see Ref. 1Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar). Nine α subunit isoforms (Nav1.1–Nav1.9) and a tenth related protein (Nax) have been identified in mammals (2Catterall W.A. Goldin A.L. Waxman S.G. Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (1060) Google Scholar). The α subunit of Navs consists of four homologous domains (DI–DIV) which compose the ion conduction pore and the voltage sensors, allowing sodium flux into the cell upon membrane depolarization and channel activation (Fig. 1B). The cytoplasmic loop linking DIII and DIV contains the gate, which binds to and occludes the cytoplasmic face of the channel pore during fast inactivation (3Patton D.E. West J.W. Catterall W.A. Goldin A.L. Neuron. 1993; 11: 967-974Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 4West J.W. Patton D.E. Scheuer T. Wang Y. Goldin A.L. Catterall W.A. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 10910-10914Crossref PubMed Scopus (661) Google Scholar). For the cardiac channel Nav1.5, it has been proposed that interaction between the DIII-DIV loop and the C-terminal domain of the α subunit stabilizes the inactivated state of the channel (5Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (125) Google Scholar). voltage-gated sodium channel fibroblast growth factor FGF receptor FGF homologous factor axon initial segment surface plasmon resonance green fluorescent protein. voltage-gated sodium channel fibroblast growth factor FGF receptor FGF homologous factor axon initial segment surface plasmon resonance green fluorescent protein. The functional importance of the cytoplasmic C-terminal domain in Nav physiology is evidenced by the fact that mutations at this region (Fig. 1B) have been linked to epilepsy, autism, cardiac arrhythmias, and skeletal muscle disease (6George Jr., A.L. J. Clin. Invest. 2005; 115: 1990-1999Crossref PubMed Scopus (286) Google Scholar). The channel C-terminal domain has been shown to contain binding sites for channel regulatory proteins (7Abriel H. Kass R.S. Trends Cardiovasc. Med. 2005; 15: 35-40Crossref PubMed Scopus (134) Google Scholar), including fibroblast growth factor homologous factors FHF1–FHF4 (8Olsen S.K. Garbi M. Zampieri N. Eliseenkova A.V. Ornitz D.M. Goldfarb M. Mohammadi M. J. Biol. Chem. 2003; 278: 34226-34236Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 9850-9857Crossref PubMed Scopus (334) Google Scholar). Because of sequence homology, FHFs are also classified as members of the fibroblast growth factor (FGF) family using the following terminologies: FHF1 = FGF12 = iFGF12, FHF2 = FGF13 = iFGF13, FHF3 = FGF11 = iFGF11, FHF4 = FGF14 = iFGF14 (10Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar, 11Ornitz D.M. Itoh N. Genome Biology. 2001; 2 (REVIEWS3005)Crossref PubMed Google Scholar, 12Itoh N. Ornitz D.M. Dev. Dyn. 2008; 237: 18-27Crossref PubMed Scopus (321) Google Scholar). However, FHFs do not bind and activate FGF receptors (FGFRs) (8Olsen S.K. Garbi M. Zampieri N. Eliseenkova A.V. Ornitz D.M. Goldfarb M. Mohammadi M. J. Biol. Chem. 2003; 278: 34226-34236Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), and therefore, we have suggested referring to these proteins as FHFs (8Olsen S.K. Garbi M. Zampieri N. Eliseenkova A.V. Ornitz D.M. Goldfarb M. Mohammadi M. J. Biol. Chem. 2003; 278: 34226-34236Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 13Goldfarb M. Cytokine Growth Factor Rev. 2005; 16: 215-220Crossref PubMed Scopus (155) Google Scholar). For each of the four mammalian FHFs, multiple isoforms with distinct N-terminal sequences are generated through alternative promoter usage and 5′ alternative splicing (14Munoz-Sanjuan I. Smallwood P.M. Nathans J. J. Biol. Chem. 2000; 275: 2589-2597Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), giving rise to 10 FHF isoforms in humans (Fig. 4). These isoforms may differ in their subcellular localization and have been shown to differentially modulate channel function (15Lou J.Y. Laezza F. Gerber B.R. Xiao M. Yamada K.A. Hartmann H. Craig A.M. Nerbonne J.M. Ornitz D.M. J. Physiol. 2005; 569: 179-193Crossref PubMed Scopus (146) Google Scholar, 16Rush A.M. Wittmack E.K. Tyrrell L. Black J.A. Dib-Hajj S.D. Waxman S.G. Eur. J. Neurosci. 2006; 23: 2551-2562Crossref PubMed Scopus (66) Google Scholar). FHFs are predominantly expressed in developing and adult neurons of the central and peripheral nervous systems (9Smallwood P.M. Munoz-Sanjuan I. Tong P. Macke J.P. Hendry S.H. Gilbert D.J. Copeland N.G. Jenkins N.A. Nathans J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 9850-9857Crossref PubMed Scopus (334) Google Scholar, 17Hartung H. Feldman B. Lovec H. Coulier F. Birnbaum D. Goldfarb M. Mech. Dev. 1997; 64: 31-39Crossref PubMed Scopus (101) Google Scholar, 18Muñoz-Sanjuán I. Fallon J.F. Nathans J. Mech. Dev. 2000; 95: 101-112Crossref PubMed Scopus (13) Google Scholar) and co-localize with Navs at axon initial segments (AIS) and nodes of Ranvier (15Lou J.Y. Laezza F. Gerber B.R. Xiao M. Yamada K.A. Hartmann H. Craig A.M. Nerbonne J.M. Ornitz D.M. J. Physiol. 2005; 569: 179-193Crossref PubMed Scopus (146) Google Scholar, 19Goldfarb M. Schoorlemmer J. Williams A. Diwakar S. Wang Q. Huang X. Giza J. Tchetchik D. Kelley K. Vega A. Matthews G. Rossi P. Ornitz D.M. D'Angelo E. Neuron. 2007; 55: 449-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 20Wittmack E.K. Rush A.M. Craner M.J. Goldfarb M. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2004; 24: 6765-6775Crossref PubMed Scopus (113) Google Scholar). Missense or frameshift mutations in the fhf4 gene have been identified in patients with inherited spinocerebellar ataxia (21Brusse E. de Koning I. Maat-Kievit A. Oostra B.A. Heutink P. van Swieten J.C. Movement Disorders. 2006; 21: 396-401Crossref PubMed Scopus (86) Google Scholar, 22Dalski A. Atici J. Kreuz F.R. Hellenbroich Y. Schwinger E. Zühlke C. Eur. J. Hum. Genet. 2005; 13: 118-120Crossref PubMed Scopus (78) Google Scholar, 23van Swieten J.C. Brusse E. de Graaf B.M. Krieger E. van de Graaf R. de Koning I. Maat-Kievit A. Leegwater P. Dooijes D. Oostra B.A. Heutink P. Am. J. Hum. Genet. 2003; 72: 191-199Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Syndromic and nonspecific forms of X-linked mental retardation have been mapped to the fhf2 gene locus, making the fhf2 gene a positional candidate gene for these disorders (24Ferrero G.B. Gebbia M. Pilia G. Witte D. Peier A. Hopkin R.J. Craigen W.J. Shaffer L.G. Schlessinger D. Ballabio A. Casey B. Am. J. Hum. Genet. 1997; 61: 395-401Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 25Gecz J. Baker E. Donnelly A. Ming J.E. McDonald-McGinn D.M. Spinner N.B. Zackai E.H. Sutherland G.R. Mulley J.C. Hum. Genet. 1999; 104: 56-63Crossref PubMed Scopus (70) Google Scholar, 26Gedeon A.K. Glass I.A. Connor J.M. Mulley J.C. Am. J. Med. Genet. 1996; 64: 121-124Crossref PubMed Scopus (24) Google Scholar, 27Malmgren H. Sundvall M. Dahl N. Gustavson K.H. Annerén G. Wadelius C. Steén-Bondeson M.L. Pettersson U. Am. J. Hum. Genet. 1993; 52: 1046-1052PubMed Google Scholar, 28Shiloh Y. Litvak G. Ziv Y. Lehner T. Sandkuyl L. Hildesheimer M. Buchris V. Cremers F.P. Szabo P. White B.N. Am. J. Hum. Genet. 1990; 47: 20-27PubMed Google Scholar). Gene knock-out studies have also implicated FHFs in development and function of the nervous system. In frog embryos, fhf2 gene knock-out suppresses neuronal differentiation (29Nishimoto S. Nishida E. J. Biol. Chem. 2007; 282: 24255-24261Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Fhf4-null mice show sensorimotor and cognitive abnormalities that resemble symptoms seen in ataxia patients carrying fhf4 mutations (30Wang Q. Bardgett M.E. Wong M. Wozniak D.F. Lou J. McNeil B.D. Chen C. Nardi A. Reid D.C. Yamada K. Ornitz D.M. Neuron. 2002; 35: 25-38Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 31Wozniak D.F. Xiao M. Xu L. Yamada K.A. Ornitz D.M. Neurobiol. Dis. 2007; 26: 14-26Crossref PubMed Scopus (75) Google Scholar). Impaired excitability of cerebellar granule and Purkinje neurons in fhf4 knock-out mice (19Goldfarb M. Schoorlemmer J. Williams A. Diwakar S. Wang Q. Huang X. Giza J. Tchetchik D. Kelley K. Vega A. Matthews G. Rossi P. Ornitz D.M. D'Angelo E. Neuron. 2007; 55: 449-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 32Shakkottai V.G. Xiao M. Xu L. Wong M. Nerbonne J.M. Ornitz D.M. Yamada K.A. Neurobiol. Dis. 2009; 33: 81-88Crossref PubMed Scopus (89) Google Scholar) may contribute to the ataxia phenotype. The fhf4 null mice also display impaired hippocampal synaptic transmission and plasticity (33Xiao M. Xu L. Laezza F. Yamada K. Feng S. Ornitz D.M. Mol. Cell. Neurosci. 2007; 34: 366-377Crossref PubMed Scopus (64) Google Scholar). In mice deficient for both fhf4 and fhf1, the ataxia phenotype is aggravated (19Goldfarb M. Schoorlemmer J. Williams A. Diwakar S. Wang Q. Huang X. Giza J. Tchetchik D. Kelley K. Vega A. Matthews G. Rossi P. Ornitz D.M. D'Angelo E. Neuron. 2007; 55: 449-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The excitability of cerebellar granule neurons in fhf1/fhf4-null mice is more severely impaired than in fhf4-null mice and is due to a substantial hyperpolarizing shift in the voltage dependence of sodium channel inactivation and slower recovery of channels from fast inactivation (19Goldfarb M. Schoorlemmer J. Williams A. Diwakar S. Wang Q. Huang X. Giza J. Tchetchik D. Kelley K. Vega A. Matthews G. Rossi P. Ornitz D.M. D'Angelo E. Neuron. 2007; 55: 449-463Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Reciprocally, transfection of FHF4 proteins into primary hippocampal neurons induces a depolarizing shift in the voltage dependence of sodium channel inactivation (15Lou J.Y. Laezza F. Gerber B.R. Xiao M. Yamada K.A. Hartmann H. Craig A.M. Nerbonne J.M. Ornitz D.M. J. Physiol. 2005; 569: 179-193Crossref PubMed Scopus (146) Google Scholar). Previously, we solved a crystal structure of FHF1B and demonstrated that FHFs are functionally distinct from FGFs even though FHFs share remarkable structural homology with FGFs (8Olsen S.K. Garbi M. Zampieri N. Eliseenkova A.V. Ornitz D.M. Goldfarb M. Mohammadi M. J. Biol. Chem. 2003; 278: 34226-34236Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Here, we present a crystal structure of FHF2A, which enabled us to define a conserved surface on FHFs required for binding to the C-terminal domain of Navs and for the modulation of channel inactivation. The FHF2A structure also reveals how a FHF invariant secondary structure in the C-terminal region of FHFs contributes to the inability of FHFs to bind FGFRs, further substantiating FHFs as functionally distinct from FGFs. Surface plasmon resonance (SPR) experiments were performed on a Biacore 2000 instrument (Biacore AB), and FHF-channel interactions were studied at 25 °C. To analyze FHF1 binding to the C-terminal domain of Nav α subunit isoforms, FHF1A1–243 and FHF1B1–181 (Fig. 1A) were immobilized by amine coupling on two flow channels of a research grade CM5 chip (Biacore AB; ∼91 fmol/mm2 of flow channel). To control for nonspecific binding, FGF21–155 was coupled to the control flow channel of the chip (∼84 fmol/mm2). Increasing concentrations of the cytoplasmic C-terminal tail of Nav1.5 (Nav1.5CT) in HBS-EP buffer (10 mm HEPES-NaOH, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% (v/v) polysorbate 20) were injected over the chip at a flow rate of 50 μl min−1. At the end of each protein injection (180 s), HBS-EP buffer (50 μl min−1) was flowed over the chip to monitor dissociation for 180 s. The chip surface was then regenerated by injecting 50 μl of 2.0 m NaCl in 10 mm sodium acetate, pH 4.5. The data were processed with BiaEvaluation software (Biacore AB). For each Nav1.5 injection, the nonspecific responses from the FGF2 control flow channel were subtracted from the responses recorded for the FHF1 flow channel. Maximal equilibrium responses were plotted against the concentrations of Nav1.5CT (Fig. 2, A and B), and from the fitted saturation binding curve the equilibrium dissociation constant (KD) was calculated. Fitted binding curves were judged to be accurate based on the distribution of the residuals (even and near zero) and χ2 ( 5 gigaohms. Voltage clamp commands and current recordings were performed with an Axopatch 200B amplifier and Digidata 1322 analog-digital interface using pClamp9 software (Axon Instruments). Cells were held at a membrane voltage of −110 mV. To measure voltage dependence of sodium channel fast inactivation, membrane voltage was stepped from −110 mV to conditioning voltages (between −110 and −20 mV, at 5-mV intervals) for 60 ms followed by further depolarization to 0 mV. Peak inward sodium currents were converted to fraction of conductance available (not inactivated), and these values for each cell were used to calculate membrane voltage of half-maximal inactivation (V1/2inact) by fitting to the Boltzmann equation, ƒ(Vm) = 1/{1 + e([Vm − V1/2inact]/k}. Statistical significance of differences in fitted parameter values between Neuro2A cells co-expressing GFP and FHF proteins and untransfected cells was calculated by the two-tailed Student's t test. Similarly, statistical significance of differences in AIS enrichment index between neurons expressing wild-type FHFs and neurons expressing mutant FHFs were determined using Student's t test. The following methodologies can be found in the supplemental material: purification of FHF and channel proteins, analysis of FHF2A crystal by mass spectrometry, and analysis of FHF1B-Nav1.6 interaction by SPR spectroscopy. Interaction between FHFs and Navs has been shown using yeast two-hybrid, far-Western blot, and co-immunoprecipitation assays (15Lou J.Y. Laezza F. Gerber B.R. Xiao M. Yamada K.A. Hartmann H. Craig A.M. Nerbonne J.M. Ornitz D.M. J. Physiol. 2005; 569: 179-193Crossref PubMed Scopus (146) Google Scholar, 16Rush A.M. Wittmack E.K. Tyrrell L. Black J.A. Dib-Hajj S.D. Waxman S.G. Eur. J. Neurosci. 2006; 23: 2551-2562Crossref PubMed Scopus (66) Google Scholar, 20Wittmack E.K. Rush A.M. Craner M.J. Goldfarb M. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2004; 24: 6765-6775Crossref PubMed Scopus (113) Google Scholar, 39Liu C.J. Dib-Hajj S.D. Waxman S.G. J. Biol. Chem. 2001; 276: 18925-18933Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 40Liu C.J. Dib-Hajj S.D. Renganathan M. Cummins T.R. Waxman S.G. J. Biol. Chem. 2003; 278: 1029-1036Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). None of these experimental approaches, however, can unambiguously show whether FHF and channel interact directly. Thus, we used SPR spectroscopy as well as size-exclusion chromatography to examine whether FHF-channel binding is direct. To this end, we expressed and purified the two human full-length splice isoforms of FHF1, FHF1A1–243 and FHF1B1–181 (Fig. 1A), and C-terminal fragments of Nav1.1 (Glu-1787 to Arg-1912; Nav1.1CT), Nav1.2 (Glu-1777 to Gln-1923; Nav1.2CT), Nav1.5 (Glu-1773 to Arg-1919; Nav1.5CT), Nav1.6 (Glu-1765 to Arg-1911; Nav1.6CT), and Nav1.9 (Glu-1585 to Glu-1729; Nav1.9CT) (Fig. 1B). These Nav fragments were designed based on previous studies showing that this channel region can bind FHF1B (39Liu C.J. Dib-Hajj S.D. Waxman S.G. J. Biol. Chem. 2001; 276: 18925-18933Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 40Liu C.J. Dib-Hajj S.D. Renganathan M. Cummins T.