A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m605473200
ISSN1083-351X
AutoresIan W. Glaaser, John R. Bankston, Huajun Liu, Michihiro Tateyama, Robert S. Kass,
Tópico(s)Analytical Chemistry and Sensors
ResumoPerturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it. Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it. Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1Hubner C.A. Jentsch T.J. Hum. Mol. Genet. 2002; 11: 2435-2445Crossref PubMed Scopus (182) Google Scholar). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na+ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2George Jr., A.L. Epilepsy Curr. 2004; 4: 65-70Crossref PubMed Scopus (44) Google Scholar, 3Jurkat-Rott K. Lerche H. Lehmann-Horn F. J. Neurol. 2002; 249: 1493-1502Crossref PubMed Scopus (108) Google Scholar, 4Kass R.S. Moss A.J. J. Clin. Invest. 2003; 112: 810-815Crossref PubMed Scopus (121) Google Scholar). Clinical consequences of inherited mutations that disrupt Na+ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins. In the heart, Na+ channels (NaV1.5) 4The abbreviations used are: NaV1.5, voltage-gated sodium channel isoform 1.5; GST, glutathione S-transferase; ISUS, sustained current; WT, wild type; TTX, tetrodotoxin; HEK, human embryonic kidney. primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for NaV1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5Clancy C.E. Kass R.S. Physiol. Rev. 2005; 85: 33-47Crossref PubMed Scopus (81) Google Scholar), and in most cases, these inherited mutations disrupt channel inactivation. Fast inactivation of Na+ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization (6Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar). Inherited mutations of the III/IV linker in the cardiac Na+ channel can disrupt fast inactivation, resulting in sustained current (ISUS), which can cause long QT syndrome variant 3 (5Clancy C.E. Kass R.S. Physiol. Rev. 2005; 85: 33-47Crossref PubMed Scopus (81) Google Scholar). However, the NaV1.5 COOH terminus also has been shown to play a role in inactivation both through chimeric studies (7Mantegazza M. Yu F.H. Catterall W.A. Scheuer T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15348-15353Crossref PubMed Scopus (108) Google Scholar), through the characterization of several disease-linked mutations found in the C terminus (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11An R.H. Wang X.L. Kerem B. Benhorin J. Medina A. Goldmit M. Kass R.S. Circ. Res. 1998; 83: 141-146Crossref PubMed Scopus (171) Google Scholar), and by direct biochemical evidence for COOH terminus interactions with the cytoplasmic peptide that links domains III and IV of the α subunit (III-IV linker) (12Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (127) Google Scholar, 13Kim 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 (151) Google Scholar). Here we have tested the hypothesis that preservation of COOH terminus structure may also be critically important to NaV1.5 channel inactivation. Previously, we generated a structural model of the NaV1.5 C terminus based on homology to the amino-terminal lobe of calmodulin (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The model predicts six α-helices (H1-H6), the first four forming two EF-hand pairs. EF hands are helix-loop-helix motifs that typically, although not always, bind Ca2+ in the loops between helices and generally occur in pairs. One helix from each EF-hand pair is predicted to form interhelical contacts with a helix from the opposite EF hand, H1 with H4 and H2 with H3. In the present experiments, we focus on a possible role of the putative interface between H1 and H4 in stabilizing the COOH terminus structure and, in turn, in the control of channel inactivation. The predicted interface in NaV1.5 was initially of interest to us not only because several naturally occurring mutations predicted to be near it disrupt inactivation and cause multiple types of cardiac arrhythmias (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), but also because mutations in similar regions of a brain sodium channel isoform (NaV1.1) have also been reported to be linked to inherited epilepsies (15Fujiwara T. Sugawara T. Mazaki-Miyazaki E. Takahashi Y. Fukushima K. Watanabe M. Hara K. Morikawa T. Yagi K. Yamakawa K. Inoue Y. Brain. 2003; 126: 531-546Crossref PubMed Scopus (277) Google Scholar, 16Nabbout R. Gennaro E. Dalla Bernardina B. Dulac O. Madia F. Bertini E. Capovilla G. Chiron C. Cristofori G. Elia M. Fontana E. Gaggero R. Granata T. Guerrini R. Loi M. La Selva L. Lispi M.L. Matricardi A. Romeo A. Tzolas V. Valseriati D. Veggiotti P. Vigevano F. Vallee L. Dagna Bricarelli F. Bianchi A. Zara F. Neurology. 2003; 60: 1961-1967Crossref PubMed Scopus (231) Google Scholar, 17Spampanato J. Kearney J.A. de Haan G. McEwen D.P. Escayg A. Aradi I. MacDonald B.T. Levin S.I. Soltesz I. Benna P. Montalenti E. Isom L.L. Goldin A.L. Meisler M.H. J. Neurosci. 2004; 24: 10022-10034Crossref PubMed Scopus (142) Google Scholar). However, the importance of this interface may be more general than its role in sodium channel function, because in structures for EF hand proteins there are extensive side chain interactions between helices analogous to the first (H1) and fourth (H4) predicted helices in the NaV1.5 C terminus (see, on the World Wide Web, structbio.vanderbilt.edu/cabp_database/struct/cmaps/cmap_list.html). In addition, hydrophobic residues are conserved at this interface among EF-hand proteins (18Kretsinger R.H. Nat. Struct. Biol. 1996; 3: 12-15Crossref PubMed Scopus (49) Google Scholar, 19Linse S. Voorhies M. Norstrom E. Schultz D.A. J. Mol. Biol. 2000; 296: 473-486Crossref PubMed Scopus (24) Google Scholar). Alignment of the first four helices of all voltage-gated sodium channels demonstrates significant homology in H1 and H4, whereas H2 and H3 are not well conserved among these channels (see supplemental data). We thus focused our experiments on possible interactions between helices H1 and H4. Our results indicate that mutation of hydrophobic residues integral to the H1/H4 interface disrupts protein stability and markedly alters channel inactivation, providing evidence that stabilization of the COOH terminus structure via the H1/H4 hydrophobic interface is necessary to preserve physiologically essential inactivation of the NaV1.5 channel. Computational Analysis—A homology model was generated as described previously (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Alignments were performed using ClustalX (20Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35918) Google Scholar) with default parameters. Molecular Biology and Tissue Culture—Site-directed mutagenesis for electrophysiological studies was carried out on NaV1.5 in pcDNA3.1 (Invitrogen). Mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocols. Mutations were confirmed by DNA sequencing. Wild type NaV1.5 and NaV1.5 mutants were transiently transfected with β1 subunits into HEK 293 cells using Lipofectamine (Invitrogen) as previously described (21Tateyama M. Liu H. Yang A.S. Cormier J.W. Kass R.S. Biophys. J. 2004; 86: 1843-1851Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The plasmid used to grow proteins for fluorescence experiments was generated using QuikChange mutagenesis on a previously generated plasmid of the NaV1.5 COOH terminus in the pGEX vector (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The predicted unstructured proximal region (residues 1773-1785) was deleted using QuikChange reactions, and a stop codon was inserted following the residue at 1863. The resulting construct (NaV1.5 EF) contained those residues predicted to form the EF hands (residues 1786-1863) in the pGEX vector with a thrombin cleavage site following the coding region for GST and preceding residues 1786-1863. Electrophysiology—Wild type and mutant human sodium channel α subunits were co-expressed with human β1 subunits in HEK 293 cells, and currents were measured with whole cell patch clamp procedures as previously described (12Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (127) Google Scholar, 21Tateyama M. Liu H. Yang A.S. Cormier J.W. Kass R.S. Biophys. J. 2004; 86: 1843-1851Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In brief, whole cell Na+ current was recorded at room temperature (22 °C) using the following solutions. The internal solution contained aspartic acid (50 mmol/liter), CsCl (60 mmol/liter), Na2-ATP (5 mmol/liter), EGTA (11 mmol/liter), HEPES (10 mmol/liter), CaCl2 (1 mmol/liter), and MgCl2 (1 mmol/liter), with pH 7.4 adjusted with CsOH. The external solution contained NaCl (130 mmol/liter), CaCl2 (2 mmol/liter), CsCl (5 mmol/liter), MgCl2 (1.2 mmol/liter), HEPES (10 mmol/liter), and glucose (5 mmol/liter), with pH 7.4 adjusted with CsOH. The voltage dependence of inactivation was determined after application of conditioning pulses (500 ms) applied once every 2 s to a series of voltages followed by a test pulse (20 ms) to voltages from -130 to -20 mV. In experiments designed to measure the voltage dependence of activation, external Na+ was reduced to 30 mm using n-methyl-glucamine as an Na+ substitute. Current was measured using test pulses (40 ms) from a holding potential of -100 mV to voltages ranging from -80 to +75 mV. Persistent Na+ channel current (ISUS) was measured as the tetrodotoxin (TTX; 30 μm)-sensitive current measured at 150 ms (Tyr1795 constructs) or 200 ms (all other constructs) during depolarization to -10 mV. Unless otherwise specified, the holding potential was -100 mV. ISUS was normalized to peak TTX-sensitive Na+ channel current measured at -10 mV and plotted as percentage of peak current in relevant figures. Membrane currents were measured using whole cell patch clamp procedures, with Axopatch 200B amplifiers (Axon Instruments, Foster City, CA). Capacity current and series resistance compensation were carried out using analog techniques according to the amplifier manufacturer (Axon Instruments, Foster City, CA). PClamp8 (Axon Instruments) was used for data acquisition and initial analysis. Data are represented as mean values ± S.E. Protein Expression and Purification—Fusion proteins were transformed in BL21 (DE3) cells (Stratagene). Cells were grown to an A600 of ∼0.6, and then expression was induced with the addition of isopropyl-d-1-thiogalactopyranoside and shaking for 72 h at 16 °C. After induction, the cells were harvested and resuspended in 20 mm Tris-Cl, 100 mm NaCl, pH 7.4, supplemented with EDTA-free protease inhibitor tablets (Roche Applied Science), DNase, MgCl2, and lysozyme. Following incubation at room temperature, the samples were sonicated, and the lysates were cleared by ultracentrifugation. The NaV1.5 EF-GST fusion proteins were further purified through affinity purification on GSTrap FF columns (Amersham Biosciences). GST eluate was thrombin-digested, and proteins were then further purified by gel filtration chromatography using a Superdex 75pg 16/60 column (Amersham Biosciences). Fractions that eluted at the appropriate time relative to previously analyzed protein standards were collected for fluorescence measurements. Mass spectrometry (matrix-assisted laser desorption ionization time-of-flight) was carried out to confirm sample purity and that the protein samples were the full-length polypeptide. Samples were collected at all stages, run on 4-20% SDS-polyacrylamide precast gels (Bio-Rad), and analyzed by Coomassie Blue staining. Protein concentration for the NaV1.5 EF was determined by absorbance at 280 nm using an extinction coefficient of 8370 m-1 cm-1. Fluorescence Spectroscopy—Fluorescence spectra were obtained on a PTI QuantaMaster spectrofluorometer in a 2-ml quartz cuvette (Hellma). Protein samples were at a concentration of 5 μm in buffer containing 20 mm Tris-Cl, 100 mm NaCl, pH 7.4, or the denatured protein in the same buffer plus urea at a concentration of 7.6 m. Intrinsic tryptophan fluorescence was excited at λex = 295 nm and monitored for fluorescence emission between 295 and 395 nm. The fluorescence contributions of the buffer, urea, and acrylamide were subtracted from the total fluorescence. Fluorescence quenching data were collected with the sequential addition of the 5 m acrylamide as the quencher. Stern-Vollmer plots were constructed according to the Stern-Vollmer equation, Fo/F=1+KSV[Q](Eq. 1) where Fo and F are the fluorescence intensities in the absence and presence of the quencher acrylamide at concentration [Q], and KSV is the Stern-Vollmer constant. Analysis of Experimental Data—Analysis was carried out in Excel (Microsoft), Origin 7.0 (Microcal Software, Northampton, MA), and programs written in Matlab (The Mathworks, Natick, MA). Data are represented as mean ± S.E. Statistical significance was determined using an unpaired Student's t test; p < 0.05 was considered statistically significant. Computational Analysis of NaV1.5 COOH Terminus Predicts a Hydrophobic Interface—We previously generated a structural model of the NaV1.5 COOH terminus based on homology to the CaM amino-terminal lobe (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The model predicts a structured proximal region containing six α-helices (H1-H6), in which the first four helices are predicted to form two EF-hand pairs. One helix from each of these EF hands is predicted to form an interface with a helix from the opposite hand pair, H1 with H4 and H2 with H3 (Fig. 1A). Alignments using ClustalX (20Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35918) Google Scholar) with default parameters predict significant structural homology between the first four predicted helices of the NaV1.5 COOH terminus and helices from NMR and x-ray crystallographic studies of EF hand proteins (Fig. 1B). Whereas residues from all of the helices in paired EF hand motifs make contributions to a hydrophobic core, a cluster of hydrophobic residues, including several that are similar among EF hand proteins, is predicted at the interface between H1 and H4 (Fig. 2A, residues marked with asterisks below). We thus focused on possible interactions between these two helices and used the predictions of the model as well as what appears to be conservation of critical hydrophobic residues as a guide in determining mutations that might be expected to perturb the interface and used inactivation gating to assay the effects of these perturbations on channel function.FIGURE 2Conservation of predicted hydrophobic interface between the first (H1) and fourth (H4) helices of NaV1.5 CT and EF-hand proteins. A, sequence alignment of predicted structural regions examined in this study. Shown are the predicted first and fourth helices. Asterisks below the alignment indicate hydrophobic residues conserved among aligned EF-hand proteins. B, structural model of NaV1.5 CT showing the first and fourth predicted helices. Side chains for residues examined in this study (Tyr1795, Trp1798, Ile1853, Leu1854) are shown as stick models.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Substitution with Nonaromatic Residues at a Disease-linked Locus Alters Inactivation—One clue for the importance of this interface to sodium channel function is provided by the fact that multiple inherited mutations of a single residue Tyr1795, located near the interface (Fig. 3B), alter sodium channel inactivation and cause congenital cardiac arrhythmias (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We thus first focused on this locus. Mutation of this residue to glutamate (Y1795E) alters channel inactivation, as reflected in an increase in sustained channel activity. Current traces are illustrated in Fig. 3A, and the mutation-induced increase in sustained current (ISUS) in response to prolonged depolarization (right traces) is evident when compared with traces from wild type (WT) channels (left traces). Space-filling models of the side chain substitution at this residue within the model (Fig. 3B) illustrate the relative difference in size of the side chains of these amino acids (197 Å3 for tyrosine and 134.8 Å3 for glutamate (22Zamyatnin A.A. Prog. Biophys. Mol. Biol. 1972; 24: 107-123Crossref PubMed Scopus (463) Google Scholar)). Replacement of Tyr1795 by Glu increases ISUS in a manner that is very similar to previously reported changes in ISUS caused by the naturally occurring mutation Y1795C (10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) (Fig. 3C and Table 1) that results in a slightly larger change in the size of the residue side chain (108.5 Å3 for the cysteine side chain (22Zamyatnin A.A. Prog. Biophys. Mol. Biol. 1972; 24: 107-123Crossref PubMed Scopus (463) Google Scholar)). We thus asked whether mutation-altered inactivation correlated with possible structural alteration of the H1/H4 interface and constructed a series of mutations to test this hypothesis (Fig. 3C). Substitution of Y1795 by either nonpolar resides (Y1795A) or nonaromatic, charged residues (Y1795E, Y1795R), in addition to small nucleophilic residues (Y1795C, Y1795S), significantly increased ISUS (Fig. 3C). However, substitution with other aromatic residues (Y1795F or Y1795W) showed no significant alteration in persistent current. Substitution with a total of eight residues was carried out and confirmed significant differences in ISUS only between channels with aromatic (including WT channels) and nonaromatic residues (Fig. 3C and Table 1) with the exception of the Y1795H mutation. The Y1795H mutation, which has been linked to Brugada syndrome (10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), results in an increase in ISUS despite its aromatic nature (Fig. 3C and Table 1). This can be attributed to the at least partial positive charge that histidine would be expected to have in the cytoplasm. Thus, our data suggest that there is a spatial and hydrophobic importance to residues predicted to be near the interface.TABLE 1Summary of mutations Sustained sodium current were measured in response to prolonged depolarization. Voltages of half-activation and inactivation were calculated from the average of fits, using the Boltzmann function, to the experimental data from each cell (see "Materials and Methods"). Values are reported as the mean ± S.E. The number in parentheses indicates the number of experiments.ConstructIsusActivation V½Inactivation V½WT0.07 ± 0.01 (14)–25.1 ± 0.8 (5)–71.6 ± 1.7 (7)Y1795E0.24 ± 0.04ap < 0.05 (6)–21.3 ± 1.8ap < 0.05 (6)–81.9 ± 0.9ap < 0.05 (6)Y1795R0.19 ± 0.04ap < 0.05 (8)–20.9 ± 0.5ap < 0.05 (6)–82.1 ± 1.3ap < 0.05 (6)Y1795H0.24 ± 0.04ap < 0.05 (5)–20.1 ± 2.1ap < 0.05 (6)–83.3 ± 0.7ap < 0.05 (6)Y1795A0.34 ± 0.03ap < 0.05 (8)–19.2 ± 1.5ap < 0.05 (6)–78.6 ± 1.1ap < 0.05 (6)Y1795S0.26 ± 0.02ap < 0.05 (6)–19.8 ± 1.1ap < 0.05 (6)–78.8 ± 0.7ap < 0.05 (6)Y1795C0.36 ± 0.04ap < 0.05 (14)–24.6 ± 1.8 (6)–72.1 ± 1.7 (6)Y1795F0.09 ± 0.01 (6)–25.9 ± 0.7 (6)–70.5 ± 1.3 (6)W1798E0.46 ± 0.11ap < 0.05 (4)–19.6 ± 1.8ap < 0.05 (7)–77.5 ± 0.7ap < 0.05 (4)W1798A0.27 ± 0.02ap < 0.05 (6)–24.9 ± 1.7 (9)–82.2 ± 1.4ap < 0.05 (4)W1798F0.10 ± 0.02 (4)–20.0 ± 2.1ap < 0.05 (4)–79.8 ± 0.5ap < 0.05 (5)W1798L0.07 ± 0.01 (5)–22.5 ± 0.6 (4)–83.0 ± 0.4ap < 0.05 (4)I1853E1.85 ± 0.09ap < 0.05 (14)–17.5 ± 2.4ap < 0.05 (2)–95.6 ± 3.3ap < 0.05 (4)I1853A0.06 ± 0.01 (4)–27.7 ± 2.0 (6)–71.0 ± 0.8 (6)L1854E0.18 ± 0.04ap < 0.05 (4)–23.8 ± 1.2 (2)–74.6 ± 1.7 (6)L1854A0.09 ± 0.01 (4)–28.5 ± 1.5 (6)–66.1 ± 1.7 (6)Y1795A/I1853E1.65 ± 0.10ap < 0.05 (5)–21.4 ± 1.0ap < 0.05 (6)–97.2 ± 2.3ap < 0.05 (7)a p < 0.05 Open table in a new tab The effects of the Tyr1795 mutations on sustained current primarily reflect changes in inactivation that follow channel openings, but we also detected mutation-induced changes in the voltage dependence of steady-state inactivation with only modest changes in the voltage dependence of activation (Table 1). The importance of the presence of an aromatic ring or charged residue at position 1795 in the maintenance of inactivation gating raises the possibility that spacing between helices H1 and H4, determined in part by the presence of an aromatic ring at residue 1795, may be critical to channel gating and, in turn, that a putative H1/H4 interface may play a key role in the structural integrity of the COOH terminus domain. Mutation of Trp1798 Disrupts Channel Gating—If integrity of an H1/H4 interface is key to control of inactivation, mutations of other hydrophobic residues predicted to form the interface would be expected to cause similar or more severe alteration in gating (inactivation), depending on the importance of the mutated residue to the integrity of the interface. We thus systematically studied the functional consequences of mutation of additional residues predicted to be within (Fig. 2B), and possibly critical to, the interhelical interface: Trp1798 (helix 1) and Ile1853 and Leu1854 (helix 4). The functional consequences of the Trp1798 mutations are very similar to those that accompany mutation of Tyr1795, affecting inactivation with relatively minor effects on channel activation (Table 1). Introduction of nonaromatic residues (W1798A and W1798E) produced significant increases in ISUS (Fig. 4B) with accompanying negative shifts in steady-state inactivation (Fig. 4C). Similar to changes made at residue 1795, conservation of the aromatic ring at residue 1798 (W1798F) did not alter ISUS, but, in contrast with the Y1795F mutation, did shift steady-state inactivation. Because Trp1798 is predicted to be more integral to the putative hydrophobic interface than Tyr1795, we also replaced the native tryptophan by a leucine, which is a large hydrophobic, but not aromatic, residue. We found that the W1798L mutation did not increase ISUS (Fig. 4B). However, this mutation, similar to the W1798F mutation, did produce a negative shift in the voltage dependence of steady state in activation (Table 1). Mutations of Hydrophobic Residues on the Partner Helix Alter Inactivation—We next tested two H4 residues predicted to be critical to the interface within the framework of our computational model: an isoleucine at residue 1853 (Ile1853) and a leucine at residue 1854 (Leu1854). Residues at similar loci on EF-hand proteins are conserved hydrophobic residues (Fig. 2A). Based on the linear sequence of the protein, one might expect similar effects when either residue is mutated. However, the modeling of the protein structure places residue Ile1853 at a location that is more critical to the putative interhelical interface and thus predicts that mutation of Ile1853 may have a greater impact on the hydrophobic interface than mutation of residue Leu1854. Our functional experimental data support the predictions of the model. We find that mutation of each of these residues has marked consequences on channel gating; however, mutation of Ile1853 causes much greater disruption of inactivation, as reflected in increased ISUS as well as the voltage dependence of steady-state inactivation, than comparable mutation of residue Leu1854. Replacement of Leu1854 by a hydrophilic glutamate residue (L1854E), but not a nonpolar alanine residue (L1854A), results in a small, but significant, increase in ISUS with modest effects on steady-state inactivation (Fig. 5, A, C, and D). Similarly, mutation of residue I1853 to a nonpolar residue (I1853A) did not have significant effects of inactivation, but mutation to the polar residue glutamate (I1853E) results in a dramatic increase in ISUS as well as marked changes in the voltage dependence of steady-state inactivation (Fig. 5, A, C, and D). ISUS recorded for the I853
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