Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5

The voltage-gated sodium channel NaV1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of NaV1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of NaV1.5. The voltage-gated sodium channel NaV1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of NaV1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of NaV1.5. The cardiac voltage-gated sodium channel NaV1.5 mediates the voltage-dependent sodium ion permeability of excitable membranes. NaV1.5 is responsible for the initial upstroke of the action potential in the electrocardiogram. Assuming opened or closed conformations in response to the voltage difference across the membrane, the protein allows Na+ ions to pass in accordance with their electrochemical gradient. Upon repeated stimulation, channels convert to the third state known as the inactive state. Channels must pass from the inactive to the closed state before they can be opened again. The structure of the channel is dominated by four membrane spanning domains (DI, DII, DIII, DIV), each containing six transmembrane helices linked by intracellular loops. The loop between DIII and DIV is of particular interest here because it is involved in the fast-inactivation of the channel (1Yu F.H. Catterall W.A. Genome Biology.http://genomebiology.com/2003/4/3/207Date: 2003Google Scholar). There are also substantial N- and the C-terminal domains, both located on the cytoplasmic side of the membrane. Several diseases are linked to the dysfunction of NaV1.5, such as long QT syndrome, Brugada syndrome, and idiopathic ventricular fibrillation (2Abriel H. Cabo C. Wehrens X.H. Rivolta I. Motoike H.K. Memmi M. Napolitano C. Priori S.G. Kass R.S. Circ. Res. 2001; 88: 740-745Crossref PubMed Scopus (108) Google Scholar, 3Wehrens X.H. Abriel H. Cabo C. Benhorin J. Kass R.S. Circulation. 2000; 102: 584-590Crossref PubMed Scopus (77) Google Scholar, 4Priori S.G. Napolitano C. Gasparini M. Pappone C. Della Bella P. Giordano U. Bloise R. Giustetto C. De Nardis R. Grillo M. Ronchetti E. Faggiano G. Nastoli J. Circulation. 2002; 105: 1342-1347Crossref PubMed Scopus (892) Google Scholar, 5Vatta M. Dumaine R. Antzelevitch C. Brugada R. Li H. Bowles N.E. Nademanee K. Brugada J. Brugada P. Towbin J.A. Mol. Genet. Metab. 2002; 75: 317-324Crossref PubMed Scopus (51) Google Scholar, 6Chen Q. Kirsch G.E. Zhang D. Brugada R. Brugada J. Brugada P. Potenza D. Moya A. Borggrefe M. Breithardt G. Ortiz-Lopez R. Wang Z. Antzelevitch C. O'Brien R.E. Schulze-Bahr E. Keating M.T. Towbin J.A. Wang Q. Nature. 1998; 392: 293-296Crossref PubMed Scopus (1553) Google Scholar), and some of these dysfunctions are due to mutations located in the C-terminal domain (7An R.H. Wang X.L. Kerem B. Benhorin J. Medina A. Goldmit M. Kass R.S. Circ. Res. 1998; 83: 141-146Crossref PubMed Scopus (170) Google Scholar, 8Smits J.P. Eckardt L. Probst V. Bezzina C.R. Schott J.J. Remme C.A. Haverkamp W. Breithardt G. Escande D. Schulze-Bahr E. LeMarec H. Wilde A.A. J. Am. Coll. Cardiol. 2002; 40: 350-356Crossref PubMed Scopus (329) Google Scholar, 9Petitprez S. Jespersen T. Pruvot E. Keller D.I. Corbaz C. Schlapfer J. Abriel H. Kucera J.P. Cardiovasc. Res. 2008; 78: 494-504Crossref PubMed Scopus (34) Google Scholar). Substantial evidence has accumulated showing that inactivation gating of NaV1.5 is modulated in response to changes in the level of calcium (Ca2+) in the adjacent cytosol. Ca2+-dependent effects on NaV1.5 inactivation gating have been shown to play an important role in the progression of certain arrhythmia conditions. Therefore, elucidating the mechanism for the Ca2+-dependent modulation of channel function is an important objective for understanding the basis of the Brugada and Long QT syndromes and for the long range goal of finding suitable chemotherapeutic strategies to neutralize the damaging events caused by these cardiac defects. Studies from our laboratory and others have shown that three regions of NaV1.5 as well as the ubiquitous intracellular Ca2+ sensor calmodulin are involved in generating the Ca2+-dependent effects on channel function (10Kim J. Ghosh S. Liu H. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). In addition to the DIII-DIV loop noted above, the C-terminal domain contains two regions involved in Ca2+-dependent modulation of channel gating function. The first is an EF-hand domain, a structure closely associated with intracellular calcium sensing in a wide range of proteins including calmodulin. The second region of interest is an IQ motif. IQ motifs are calmodulin (CaM) 2The abbreviations used are: CaM, calmodulin; CTD, C-terminal domain; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BME, β-mercaptoethanol; MES, 4-morpholineethanesulfonic acid; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect (NOE) spectroscopy. binding domains most commonly associated with recruitment of CaM to specific sites so that it is able to respond rapidly to calcium signals. The fact that the NaV1.5 IQ motif has important functions for both the intrinsic sensing of Ca2+ by the EF-hand domain and the recruitment of CaM led us to propose a model in which the IQ motif serves as molecular switch that couples the two Ca2+-sensing modules (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). In the present study we report the solution structure of the C-terminal domain EF-hand domain (CTD-EF) in NaV1.5 determined by NMR spectroscopy. Analysis of this structure reveals important features relating to the function of this domain and provides essential groundwork for determining why functional Ca2+ binding requires an additional 60 residues downstream from the EF-hand domain (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). The implications of these results are described with respect to the mechanism of action of the complex calcium-sensing apparatus in NaV1.5. Expression and Purification-Recombinant human CTD-EF (Glu-1773-Ser-1865) was subcloned between BamHI and EcoRI restriction sites of an in-house pBG100 plasmid (Dr. L. Mizoue, Center for Structural Biology), which is derived from pET27. To facilitate purification, this vector codes for a His6 tag and a flexible linker containing a 3C protease cleavage site. The CTD-EF construct is expressed as a 97-residue protein that contains Gly-Pro-Gly-Ser fused to its N terminus after cleavage of the His6 tag. Proteins expressed in and to was and the was of and proteins by on or The was with and or as the and in and and using The solution was for was and a affinity proteins by the with BME, and and the protein was using a to BME, and containing CTD-EF and 3C protease was This solution was The solution was a affinity to CTD-EF and the His6 tag. The was on a to and an peptide containing the IQ motif of NaV1.5 was by high and by to and molecular titration calorimetry with a in and or protein and peptide the in the was by a of of CTD-EF with an of The binding with a binding model using The was from the NMR was BME, and The protein was for structure and for titration To effects on of protein solution was a NMR and this was a NMR containing of G. J. 2006; PubMed Scopus Google Scholar). NMR NMR on and with and with of Scholar). and resonance by the of and and J. W.J. NMR and Scholar). side resonance from and of side on and To a was on an and and on a The used in was as described Zhang J. NMR. PubMed Scopus Google Scholar). The and for the and and for the with and of using a of as the of with and for well The functions in used to the and which using the model in for and well The was using the for only well in the well regions of the protein A. PubMed Scopus Google Scholar). The binding of the IQ motif peptide to CTD-EF was determined by NMR of CTD-EF as the peptide was the for of and The of the was expressed as D. R. R. J. Mol. Biol. PubMed Scopus Google Scholar). first of used P. Mol. Biol. 2004; PubMed Scopus Google Scholar). The of of was from a of In each from with of and used for the The from and using S. Res. 2006; PubMed Scopus Google with a range of The target function for by molecular in T. H. R. A. C. Wang B. J. Chem. PubMed Scopus Google using the The for of molecular was with the the from to by with a coupling for and of of to with of by of and a of The from to that of their the first The to the with the and of IQ to model for the motif complex was using C. R. J. Am. Chem. PubMed Scopus Google Scholar). The residues of the CTD-EF construct from the so as to with of the The central region of the IQ motif was as a with the N- and in a in with structure B. G. Liu J. Res. 2004; PubMed Scopus Google and of IQ motif Full Text Full Text PDF PubMed Scopus Google Mol. Biol. PubMed Scopus Google Scholar). The in the of CTD-EF using S. J. of Scholar). with a and the as for the The adjacent residues with as In the first of the an initial of was The for a second with and in The from the most with the and used for with C. J. J. Mol. Biol. 2000; PubMed Scopus Google and Res. 2004; PubMed Scopus Google through the A. V. G. S. S. S. V. M. Res. 2008; PubMed Scopus Google Scholar). of the and with the R. M. K. J. Mol. PubMed Scopus Google and The Scholar). The and of the CTD-EF was using J.A. R. J. NMR. PubMed Scopus Google Scholar). with the D. S. J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). was on the NaV1.5 and subcloned the vector for of the channel protein and protein as described M.T. Bezzina C.R. Wilde A.A. Balser J.R. Nature. 2001; PubMed Scopus Google Scholar). with or for The on with with and was and as described M.T. Bezzina C.R. Wilde A.A. Balser J.R. Nature. 2001; PubMed Scopus Google Scholar). The used are shown in the to function was to the to the membrane potential inactivation is the The solution used to to with high was used with The solution and to with To the of the commonly observed voltage-dependent inactivation was within after of the membrane. are as the using with and are in the The of the of and the of NMR are in the of the EF-hand in the NaV1.5 solution structure of was determined by multidimensional heteronuclear NMR spectroscopy. To this the and to our report (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). and resonance for the of using the of and described conformations for two of the residues on the basis of and and M. D. H. P. J. NMR. 2002; PubMed Scopus Google Scholar, K. NMR of Proteins and Google Scholar). of the of range the of four which consistent with by the J. NMR. PubMed Scopus Google to the of including and The of and with are consistent with the existence of a domain between residues and to for the and are for the residues of that the N terminus of this construct is The provides an of for the consistent with the CTD-EF as a in The structure of CTD-EF was using a that of initial using P. Mol. Biol. 2004; PubMed Scopus Google and by molecular using T. H. R. A. C. Wang B. J. Chem. PubMed Scopus Google as described L. M. Chazin W.J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). In and these well the for the first which and long range In addition to derived from and the for of and and of The of was on and molecular and is shown in with the of this is well and and molecular The structure has been to high with a of for the and for The high of the structure is by of the regions of the for CTD-EF in for from the for residues and T. H. R. A. C. Wang B. J. Chem. PubMed Scopus Google J.A. R. J. NMR. PubMed Scopus Google Scholar). in a The of CTD-EF is similar to other EF-hand domains, of two EF-hand motifs and a linker between The four helices The between the two EF-hand is residues and The loop between helices and IV is it is the residues in it and a is of other EF-hand domain loops. noted above, the first residues of the CTD-EF construct are In the of the CTD-EF only to the EF-hand domain. CTD-EF have substantial with well characterized EF-hand calcium-binding proteins such as or it have a EF-hand protein The T. Biol. PubMed Scopus (892) Google three EF-hand proteins as the most domain of and calcium-binding protein the L. C. PubMed Scopus Google EF-hand proteins as the most with of for the N terminus of CaM and for the N terminus of for and for human of these which have with respect to CTD-EF of and for EF-hand of domain of of CaM, and of The of CTD-EF to functional be CTD-EF contains a of in of of the sequence. a the protein has a and a of The is consistent with the domain as part of the Ca2+-sensing regulatory apparatus of NaV1.5. The the of the domain was using D. S. J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). CTD-EF has a of that the of CTD-EF is The substantial potential I is and be in Ca2+ binding to the NaV1.5 C-terminal region has been in a and the location of the EF-hand domain was predicted on the basis of a consensus 12-residue EF-hand calcium binding loop spanning T.L. V.N. Chazin W.J. Balser J.R. Mol. Biol. 2004; PubMed Scopus Google Scholar). from determining the structure is that the location of the EF-hand domain is shifted with respect to the of the consensus EF-hand residues is located in the first EF-hand and they I. the two Ca2+ binding in the structure of only I a consistent with ion have the of residues so it is to a functional Ca2+ binding site. I well to the consensus for a functional its is similar to a Ca2+ binding loop in the EF-hand of domain and in fact they similar The loop in was first described as binding loop S. H. K. J. PubMed Scopus Google Scholar, H. K. J. PubMed Scopus Google and was characterized as an important in the of the domains IV and of Li H. W. J. 83: PubMed Scopus Google Scholar). in CTD-EF on the of NaV1.5 was on residues in the containing the consensus EF-hand Ca2+ binding loop T.L. V.N. Chazin W.J. Balser J.R. Mol. Biol. 2004; PubMed Scopus Google Scholar). structure of the CTD-EF reveals that these mutations in I and in the Ca2+ binding I as was To the functional of the EF-hand domain in the NaV1.5 mutations have been in the Ca2+ binding I and in on of I mutations of and residues in CTD-EF on the I residues predicted to be involved in Ca2+ are here for the shown in the Ca2+ for the are shifted to the channels to the Ca2+-dependent observed in NaV1.5 that was first reported in our study T.L. V.N. Chazin W.J. Balser J.R. Mol. Biol. 2004; PubMed Scopus Google Scholar). These results the role of the NaV1.5 EF-hand domain in Ca2+-dependent regulation of channel function. CTD-EF the IQ a between I and have shown that the EF-hand domain only binds Ca2+ with high affinity in a construct 60 residues downstream to the IQ motif T.L. V.N. Chazin W.J. Balser J.R. Mol. Biol. 2004; PubMed Scopus Google Scholar). evidence been the IQ motif binds to the EF-hand domain (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). To and this isothermal titration calorimetry was used to the binding affinity of an IQ motif peptide to binding the addition of and CTD-EF to of the IQ of this to a single binding provides of and for and NMR was used to the binding of the IQ motif peptide on the structure of was by NMR of CTD-EF as the peptide was the a of by the binding of the The observed changes in the which the resonance to be from the protein to the peptide and the most binding of the these residues and their the first titration of the NMR changes the structure of CTD-EF that the peptide binds to a between I and IV The binding a of residues that to the potential in this region as well as residues present in or to I and IV The binding also two residues to I and four other residues located in IV To in understanding the of the binding the used as to of the IQ motif peptide structure was for the IQ motif peptide on both structure and the of other IQ motifs in their with EF-hand proteins Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Biol. PubMed Scopus Google Scholar). the with the peptide in of two with respect to the of the model of the complex is shown in to between residues in CTD-EF and IQ residues and The IQ peptide also contains residues and that the of the CTD-EF binding the model reveals that the IQ motif a binding between I and IV on the of CTD-EF with from both and first predicted the of an EF-hand in the C-terminal region of calcium and sodium voltage-gated channels J. Nature. PubMed Scopus Google Scholar). predicted the location of the EF-hand domain on the of a consensus 12-residue EF-hand Ca2+ binding loop between and T.L. V.N. Chazin W.J. Balser J.R. Mol. Biol. 2004; PubMed Scopus Google Scholar). of the structure of the CTD-EF construct an EF-hand domain it is from our to the EF-hand Ca2+ binding loop the a that is with Ca2+ the two Ca2+ binding in the the residues to I has the to Ca2+ and in particular has the its terminus that provides of the Ca2+ The of Ca2+ binding I is similar to a Ca2+ binding loop in domain of is also the to CTD-EF and the structure of the are similar CTD-EF and EF-hand of domain have common residues the between I and including and in which to residues and The of these residues in the to their in of the of CTD-EF is in and this to for which only a of residues in are the of the residues in CTD-EF of channels the from I and IV are the most the residues and located on and the side are the The high level of of the EF-hand domain in the IQ motif binding between I and IV an important role for CTD-EF in channel function. The EF-hand domains in and channels are adjacent to the membrane. The 97-residue construct used in this study the residues that the CTD-EF domain to the of NaV1.5 NMR that these residues are and that they a flexible linker between the EF-hand domain and the transmembrane of DIV and in to the and other within the these residues are in the which their functional propose that the of this linker is important in channels for the EF-hand domain to its with respect to the membrane as it within the CTD-EF domain have important effects on channel function. and of an between and are associated with cardiac arrhythmia syndromes H. Wehrens X.H. Benhorin J. Kerem B. Kass R.S. Circulation. 2000; 102: PubMed Scopus Google Scholar, Circulation. 2002; 105: PubMed Scopus Google and I. of residues and in in are associated with T. T. E. K. M. K. T. K. K. PubMed Scopus Google Scholar). These residues to the of the EF-hand domain of residues in NaV1.5 have also been shown to to of channel function J.R. Liu H. Tateyama M. Kass R.S. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (34) Google Scholar). mutations be to the domain and its structure to of channel function. have shown here that mutations in Ca2+ binding I the Ca2+ of inactivation it is that a is to the of this domain in NaV1.5 function. The of the structure of CTD-EF a these In we that high affinity binding of Ca2+ to the EF-hand domain is observed only in a construct that downstream to the IQ motif and evidence in of an between the EF-hand domain and the IQ motif (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). The isothermal titration calorimetry showing binding of an IQ motif peptide to the CTD-EF with an affinity of between these two is of functional that the of the IQ motif with CTD-EF be in the channel because of the high due to the binding of the IQ motif the of CTD-EF to is it provides or that the it or a that is for ion The of changes in residues located in I of CTD-EF titration with the IQ motif peptide changes in the structure in this which the second However, to elucidate the basis for the effect of IQ of a construct that both of these is The structure of CTD-EF and of binding of the IQ motif to this domain is consistent with our of a central role for the IQ motif as a molecular switch that the action of intrinsic (CTD-EF) and extrinsic (calmodulin) Ca2+ of NaV1.5 (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). results evidence in of the existence of a of that Ca2+ binding by CTD-EF and calmodulin to with the IQ motif In this of of calmodulin binds so to the IQ motif that it the IQ motif from with the which in Ca2+ binding to Ca2+ are calmodulin binds the IQ motif (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar), which the of the IQ motif to to In Ca2+ calmodulin to to other regions of NaV1.5, such as the calmodulin binding domain in the linker between DIII and DIV (11Shah V.N. Wingo T.L. Weiss K.L. Williams C.K. Balser J.R. Chazin W.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3592-3597Crossref PubMed Scopus (100) Google Scholar). These Ca2+-dependent binding sites with the IQ motif for calmodulin the of the IQ motif to with results to the that the response of NaV1.5 to intracellular Ca2+ is complex and calmodulin as well as domains within the model for the action of the Ca2+-sensing apparatus provides a for to function in and in and a understanding of the molecular basis for the Ca2+ modulation of inactivation gating of NaV1.5. and for with