The KCNQ1 (Kv7.1) COOH Terminus, a Multitiered Scaffold for Subunit Assembly and Protein Interaction
2007; Elsevier BV; Volume: 283; Issue: 9 Linguagem: Inglês
10.1074/jbc.m707541200
ISSN1083-351X
AutoresReuven Wiener, Yoni Haitin, Liora Shamgar, M. Carmen Fernández‐Alonso, Ariadna Martos, Orna Chomsky-Hecht, Germán Rivas, Bernard Attali, Joel A. Hirsch,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoThe Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0Å crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation. The Kv7 subfamily of voltage-dependent potassium channels, distinct from other subfamilies by dint of its large intracellular COOH terminus, acts to regulate excitability in cardiac and neuronal tissues. KCNQ1 (Kv7.1), the founding subfamily member, encodes a channel subunit directly implicated in genetic disorders, such as the long QT syndrome, a cardiac pathology responsible for arrhythmias. We have used a recombinant protein preparation of the COOH terminus to probe the structure and function of this domain and its individual modules. The COOH-terminal proximal half associates with one calmodulin constitutively bound to each subunit where calmodulin is critical for proper folding of the whole intracellular domain. The distal half directs tetramerization, employing tandem coiled-coils. The first coiled-coil complex is dimeric and undergoes concentration-dependent self-association to form a dimer of dimers. The outer coiled-coil is parallel tetrameric, the details of which have been elucidated based on 2.0Å crystallographic data. Both coiled-coils act in a coordinate fashion to mediate the formation and stabilization of the tetrameric distal half. Functional studies, including characterization of structure-based and long QT mutants, prove the requirement for both modules and point to complex roles for these modules, including folding, assembly, trafficking, and regulation. The KCNQ channels represent a subfamily of voltage-gated K+ (Kv) 3The abbreviations used are:Kvvoltage-gated K+ channelCaMcalmodulinLQTlong QTPBSphosphate-buffered salineICPinductively coupled plasmaWTwild typeNHSN-hydroxysulfosuccinimide. channels, whose members (Kv7.1-5) are expressed in a wide variety of tissues. These channels play a major role in brain and cardiac excitability through the modulation of the cardiac potential waveform, the regulation of action potential generation and propagation, the tuning of neuronal firing patterns, and the modulation of neurotransmitter release (1Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (695) Google Scholar, 2Robbins J. Pharmacol. Ther. 2001; 90: 1-19Crossref PubMed Scopus (358) Google Scholar). Mutations in human KCNQ genes lead to major cardiovascular and neurological disorders, such as the cardiac long QT syndrome or neonatal epilepsy (3Jentsch T.J. Hubner C.A. Fuhrmann J.C. Nat. Cell Biol. 2004; 6: 1039-1047Crossref PubMed Scopus (158) Google Scholar). voltage-gated K+ channel calmodulin long QT phosphate-buffered saline inductively coupled plasma wild type N-hydroxysulfosuccinimide. Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage-sensing domain (S1-S4) and a pore domain (S5-S6) (see Fig. 1A), likely to approximate the mammalian Kv1.2 channel α structure described by MacKinnon and co-workers (4Long S.B. Campbell E.B. Mackinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1858) Google Scholar). Often, KCNQ α is in complex with the integral membrane auxiliary subunit, known as KCNE1, IsK, or MinK. This protein alters channel properties, such as single channel conductance and activation kinetics while increasing channel density in the plasma membrane (5Abbott G.W. Goldstein S.A. Mol. Interv. 2001; 1: 95-107PubMed Google Scholar). Several structural features of the Kv7 α family members distinguish them from the larger Kv channel superfamily. In particular, their primary sequence encodes a large (300-500 residues), intracellular COOH terminus (Fig. 1A). Sequence analysis predicts four helical regions (dubbed A-D) present in all family members (6Yus-Najera E. Santana-Castro I. Villarroel A. J. Biol. Chem. 2002; 277: 28545-28553Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Helices C and D are thought to form coiled-coil assemblies. Yeast two-hybrid screens for proteins interacting with the COOH terminus revealed CaM, the ubiquitous Ca2+ sensor protein, as a binding partner (6Yus-Najera E. Santana-Castro I. Villarroel A. J. Biol. Chem. 2002; 277: 28545-28553Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Wen H. Levitan I.B. J. Neurosci. 2002; 22: 7991-8001Crossref PubMed Google Scholar, 8Shamgar L. Ma L. Schmitt N. Haitin Y. Peretz A. Wiener R. Hirsch J. Pongs O. Attali B. Circ. Res. 2006; 98: 1055-1063Crossref PubMed Scopus (163) Google Scholar). Moreover, CaM constitutively associates with the channel (6Yus-Najera E. Santana-Castro I. Villarroel A. J. Biol. Chem. 2002; 277: 28545-28553Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Wen H. Levitan I.B. J. Neurosci. 2002; 22: 7991-8001Crossref PubMed Google Scholar, 8Shamgar L. Ma L. Schmitt N. Haitin Y. Peretz A. Wiener R. Hirsch J. Pongs O. Attali B. Circ. Res. 2006; 98: 1055-1063Crossref PubMed Scopus (163) Google Scholar, 9Gamper N. Stockand J.D. Shapiro M.S. J. Neurosci. 2003; 23: 84-95Crossref PubMed Google Scholar, 10Gamper N. Li Y. Shapiro M.S. Mol. Biol. Cell. 2005; 16: 3538-3551Crossref PubMed Scopus (120) Google Scholar). Helices A and B encode the binding site for CaM, and CaM association is required for proper channel assembly and function (6Yus-Najera E. Santana-Castro I. Villarroel A. J. Biol. Chem. 2002; 277: 28545-28553Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Wen H. Levitan I.B. J. Neurosci. 2002; 22: 7991-8001Crossref PubMed Google Scholar, 9Gamper N. Stockand J.D. Shapiro M.S. J. Neurosci. 2003; 23: 84-95Crossref PubMed Google Scholar). LQT mutations that disrupt or significantly weaken the CaM interaction result in little or no complex and little channel expression in live cells (8Shamgar L. Ma L. Schmitt N. Haitin Y. Peretz A. Wiener R. Hirsch J. Pongs O. Attali B. Circ. Res. 2006; 98: 1055-1063Crossref PubMed Scopus (163) Google Scholar, 11Ghosh S. Nunziato D.A. Pitt G.S. Circ. Res. 2006; 98: 1048-1054Crossref PubMed Scopus (136) Google Scholar). Thus, CaM acts as an additional auxiliary subunit of the KCNQ channel complex. Additionally, Ca2+-CaM is a Ca2+ sensor for KCNQ1 function, transducing Ca2+ signals to stimulate IKS channels and producing a Ca2+-dependent left shift in the voltage dependence of channel activation. This Ca2+-sensitive IKS-current stimulation could increase the cardiac repolarization reserve, preventing the risk of ventricular arrhythmias. Biochemical and functional studies have identified Kv7 COOH-terminal regions important for channel tetramerization and trafficking (12Schmitt N. Schwarz M. Peretz A. Abitbol I. Attali B. Pongs O. EMBO J. 2000; 19: 332-340Crossref PubMed Scopus (141) Google Scholar, 13Schwake M. Jentsch T.J. Friedrich T. EMBO Rep. 2003; 4: 76-81Crossref PubMed Scopus (119) Google Scholar, 14Schwake M. Athanasiadu D. Beimgraben C. Blanz J. Beck C. Jentsch T.J. Saftig P. Friedrich T. J. Neurosci. 2006; 26: 3757-3766Crossref PubMed Scopus (80) Google Scholar, 15Kanki H. Kupershmidt S. Yang T. Wells S. Roden D.M. J. Biol. Chem. 2004; 279: 33976-33983Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16Maljevic S. Lerche C. Seebohm G. Alekov A.K. Busch A.E. Lerche H. J. Physiol. 2003; 548: 353-360PubMed Google Scholar) based on deletion, truncation, and mutagenesis. Little work has directly examined on a protein level the postulated structural modules and their functional correlates. What precisely is the tertiary and quaternary organization of the COOH terminus, and what does it do that distinguishes it from the other Kv channel subfamilies? Using a recombinant bacterial co-expression system (8Shamgar L. Ma L. Schmitt N. Haitin Y. Peretz A. Wiener R. Hirsch J. Pongs O. Attali B. Circ. Res. 2006; 98: 1055-1063Crossref PubMed Scopus (163) Google Scholar), we have dissected the KCNQ1 COOH terminus protein complex. Our findings suggest that the KCNQ1 COOH terminus may be divided into two parts, where the membrane proximal half is important for functional expression, folding, and gating of the channel but not oligomerization, whereas the membrane distal half directs folding, oligomerization, partner specificity, and trafficking to the plasma membrane. This COOH terminus is a multifunctional platform, employing relatively simple structural modules to assemble a channel with high specificity in an apparent hierarchical manner. For bacterial expression vectors, PCR was used to engineer BamHI and NotI restriction sites onto the specific human KCNQ1 gene fragments. PCR product was ligated into doubly digested (BamHI and NotI) modified pETDuet vector. This pETDuet vector contains a His8 tag sequence and TEV protease recognition sequence upstream to multiple cloning site I with CaM inserted into multiple cloning site II. Positive clones were identified by restriction analysis and subsequently sequenced in all DNA constructs described below. In addition, all site-directed mutagenesis was performed by the QuikChange (Stratagene) method. Altered sequences were confirmed by DNA sequencing. Subcloning of Δ Helix C—The KCNQ1 sequence in a pcDNA3 vector was used as a template for QuikChange, where primers were designed to delete helix C (residues 548-565). This modified vector then served as the template for PCR of an insert for the pETDuet vector. Subcloning of Δ Loop—Construction was in two steps. First, PCR was used to engineer BamHI and EcoRI restriction sites onto a KCNQ1 gene fragment (residues 352-386). PCR product was ligated into a doubly digested (BamHI and EcoRI), modified pETDuet vector. Then PCR was used to engineer EcoRI and NotI restriction sites onto an additional gene fragment (residues 504-622). This PCR product was ligated into doubly digested (EcoRI and NotI) modified pETDuet vector already contained the fragment encoding residues 352-386. Subcloning of COOH Terminus-GCN4-LI Chimera—Subcloning was in two steps. First, PCR was used to engineer BamHI and EcoRI restriction sites into the KCNQ1 gene fragment (residues 352-594). PCR product was ligated into doubly digested (BamHI and EcoRI) modified pETDuet vector. Then the GCN4-LI sequence (encoding IEDKLEEILSKLYHIENE-LARIKKLLG) was amplified by primers containing EcoRI and NotI sites in the 5′- and the 3′-flanking regions, respectively. GCN4-LI PCR product was ligated into doubly digested (EcoRI and NotI) modified pETDuet vector that already contained DNA encoding residues 352-594. Subsequently, the EcoRI restriction site was deleted by mutagenesis. Transformed Escherichia coli BL-21 Tuner (Novagen), containing the "RIL" Codon Plus™ plasmid (Stratagene) cells were grown at 37 °C in LB medium, containing 100 μg/ml ampicillin, and 34 μg/ml chloramphenicol. Upon reaching an A600 of 0.3, the temperature was lowered to 16 °C, and growth continued until the culture reached an A600 of 0.6. Protein expression was induced with 135 μm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested after 14 h by centrifugation, frozen, and suspended in 120 ml of lysis buffer, buffer L (300 mm NaCl, 50 mm sodium phosphate, pH 8, 1 mm phenylmethylsulfonyl fluoride). After lysis by a microfluidizer (Microfluidics), cell debris was removed by centrifugation at 20,000 × g. The soluble fraction was loaded onto a pre-equilibrated metal chelate Ni2+-CAM (Sigma) column (buffer A: 300 mm NaCl, 50 mm sodium phosphate, pH 8) at a flow rate of 1.0 ml/min. The column was washed with buffer A, containing 10 mm imidazole, until a stable base line was achieved. After elution with buffer A, supplemented with 125 mm imidazole, the protein eluate was then subjected to TEV protease in a ratio of 1:150. The proteolysis continued for 12 h. Subsequently, the sample was diluted 4-fold with 10% glycerol and loaded onto a pre-equilibrated SP-Sepharose (Amersham Biosciences) column (buffer S: 50 mm NaCl, 20 mm Tris, pH 7.5). The column was then washed with buffer S, and fractions were eluted with a shallow linear gradient of buffer S, containing 50-600 mm NaCl. Fractions were pooled and applied to a pre-equilibrated Superose 6 gel filtration column (Amersham Biosciences) with buffer F (150 mm NaCl, 20 mm Tris, pH 7.5, 1 mm dithiothreitol). The elution peak was concentrated to 3 mg ml-1 using spin concentrators (Vivascience), divided into aliquots, and flash-frozen in liquid N2. For purification of Δ helix D, Δ helices C-D, and Δ loop-CaM complexes, a Superdex-200 gel filtration column was employed instead of the Superose-6 gel filtration column. Both KCNQ1 gene fragments (residues 535-572 or 535-622, respectively) were amplified by PCR with primers containing BamHI and NotI sites in the 5′- and 3′-flanking regions, respectively. PCR product was ligated into doubly digested (BamHI and NotI) pGEX 4T-1 vector (Amersham Biosciences). Expression was as above. Cells were suspended in phosphate-buffered saline (PBS) (pH 7.4) with 1 mm phenylmethylsulfonyl fluoride. After lysis, cell debris was removed by centrifugation. The soluble fraction was loaded onto a pre-equilibrated glutathione-Sepharose column (buffer A: PBS, pH 7.4), at a flow rate of 1.0 ml/min. The column was washed with buffer A and eluted with buffer B (100 mm Tris pH 8, 100 mm NaCl, 20 mm glutathione), and the protein was applied to a pre-equilibrated Superdex 200 gel filtration column with buffer F. The elution peak was concentrated and stored as above. The selenomethionine Δ loop-CaM complex was prepared by inhibition of the methionine pathway. Inoculum was grown from a single transformed colony in 10% LB medium. The cells were pelleted, and media supernatant was removed, prior to the addition to 2 liters of New Minimal Medium, containing Kao and Michayluk vitamin solution (Sigma), 100 μg/ml ampicillin, and 34 μg/ml chloramphenicol. Upon reaching an A600 of 0.3, the temperature was lowered to 16 °C. Lysine, phenylalanine, and threonine (100 mg/liter), isoleucine, leucine, and valine (50 mg/liter), and dl-selenomethionine (50 mg/liter) were added 45 min before induction, when the culture reached an A600 of 0.6. Protein expression was induced with 135 μm isopropyl 1-thio-β-d-galactopyranoside over a 16-h period. Purification of the selenomethionine protein was similar to that of the native protein, except that 5 mm β-mercaptoethanol was added to all solutions to prevent oxidation of the selenomethionine derivative protein. The helix D synthetic peptide (residues 585-621, acetylated and amidated at the NH2 and COOH termini, respectively) was purified by reverse-phase high pressure liquid chromatography using a C18 column (Vydac) with a shallow acetonitrile gradient of 35-60% (both solvents were supplemented with 0.05% trifluoroacetic acid). Sedimentation velocity analysis of the different protein samples were carried out at 40,000 or 50,000 rpm and 5 °C (except Δ helices C-D and point mutants that were centrifuged at 10 °C) in an XL-A analytical ultracentrifuge (Beckman-Coulter Inc.) with a UV-visible optics detection system, using an An60Ti rotor and 12-mm double sector centerpieces. All of the proteins were equilibrated in 20 mm Tris-HCl, 150 mm NaCl, 0.1 mm dithiothreitol, pH 7.5, buffer except for CT-GCN4LI, which was in PBS. The sedimentation velocity runs were done at different protein concentrations ranging from 0.1 to 1.5 mg/ml. Sedimentation profiles were registered every 5 min at the appropriate wavelength (ranging from 230 to 280 nm). The sedimentation coefficient distributions were calculated by least-squares boundary modeling of sedimentation velocity data using the c(s) method (17Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar) as implemented in the SEDFIT program. The corresponding standard s-values (S20,w) were obtained from the experimental s-values upon correction for density, viscosity, and protein concentration (18van Holde K.E. Physical Biochemistry. 2nd Ed. Prentice-Hall, Englewood Cliffs, NJ1986: 110-136Google Scholar), using the SEDNTERP program (19Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). Sedimentation equilibrium experiments were carried out at multiple speeds (9000, 13,000, 22,000, 32,000, 40,000, and 43,000 rpm) and wavelengths (230, 238, 250, and 280 nm) with short columns (80 μl) using the same instrumentation and conditions as described above. In order to determine the stoichiometry of the two KCNQ-CaM complexes, the sedimentation equilibrium data of these macromolecular mixtures were analyzed assuming the linear approximation of the buoyant molecular weights (20Cole J.L. Methods Enzymol. 2004; 384: 212-232Crossref PubMed Scopus (110) Google Scholar, 21Rivas G. Stafford W. Minton A.P. Methods. 1999; 19: 194-212Crossref PubMed Scopus (77) Google Scholar), bMw,ij = ibMw,A + jbMw,B, where ij refers to the complex AiBj, and bMw,A and bMw,B are the buoyant molecular weights of pure A and pure B, respectively. In this case, the weight-average buoyant molecular masses (bMw) were determined from the sedimentation equilibrium data by fitting a single species model to the experimental data using either a MATLAB program based on the conservation of signal algorithm (22Minton A.P. Schuster T.M. Laue T.M. Modern Analytical Ultracentrifugation. Birkhauser, Boston, MA1994: 81-93Google Scholar) (kindly provided by Dr. Allen Minton, National Institutes of Health) or the HeteroAnalysis program (20Cole J.L. Methods Enzymol. 2004; 384: 212-232Crossref PubMed Scopus (110) Google Scholar); both analytical methods gave essentially the same results. The molecular masses of the single solute components (helix D peptide and CaM) were determined from the corresponding experimental buoyant values using 0.734 and 0.732 cm/g as the partial specific volumes of peptide and CaM, respectively, calculated from the amino acid composition using the SEDNTERP program. All measurements were done with an Aviv CD spectrometer model 202. Spectra were measured over the range of 260-180 nm at a scan rate of 1 nm/s. A cell with 0.1-mm path length was used. Each spectrum is an average of five scans. The raw data were corrected by subtracting the contribution of the buffer to the signal. Then data were smoothed and converted to molar ellipticity units. Protein concentration was determined using the predicted extinction coefficient at 280 nm. For melting experiments, CD was measured at 222 nm with a 1-mm path length cell. Temperature equilibrium time was 2 min, and integration time was 30 s. The CD data were scaled from 0 to 1 with respect to the initial native form and the fully unfolded form using the equation, CDscaled=CDobs-CDinitialCDfinal-CDinitial Proteins were injected onto a Superose 6 10/300 GL or Superdex 200 10/300 GL or Superdex 75 10/300 GL (Amersham Biosciences) column. Proteins were eluted with buffer F at a flow rate of 0.5, 0.7, or 0.8 ml/min for Superose 6, Superdex 200, or Superdex 75 columns, respectively. Crystals were grown in 12-16% polyethylene glycol 8000, 0.1 m Tris, pH 7-8, at 19 °C by sitting drop vapor diffusion. Equal volumes (1 μl) of frozen stock protein (15 mg/ml) were mixed with reservoir solution. Native crystals appeared after 2 weeks. Heavy atom soaks were prepared by adding 1 mm K2ReCl6 directly to drops containing crystals for 24 h. Four selenomethionine protein crystals appeared after several months. Crystals were cryoprotected by sequential dilution with reservoir solution, including 24% glycerol, and then loop mounted and flash frozen in liquid N2. Diffraction data were obtained under standard cryogenic conditions and processed with HKL (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). We executed a single wavelength anomalous diffraction experiment on the selenomethionine crystals, using the anomalous selenium peak. Heavy atom site location and experimental phases calculation was performed with CNS (24Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and SHARP (25de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar, 26Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar), respectively. Multiple isomorphous replacement with anomalous scattering from the selenium and rhenium derivative data sets against the native data set were used for experimental phase calculation. Subsequent density modification with SOLOMON (26Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1142) Google Scholar) gave electron density maps of excellent quality. An initial model was built with ARP/wARP (27Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (475) Google Scholar) and subsequently refined with Refmac5 (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) with rounds of model building. Recordings were performed using the whole cell configuration of the patch clamp technique. Signals were amplified using an Axopatch 200B patch clamp amplifier (Axon Instruments), sampled at 2 kHz and filtered at 800 Hz via a 4-pole Bessel low pass filter. Data were acquired using pClamp 8.2 software in conjunction with a DigiData 1322A interface. The patch pipettes were pulled from borosilicate glass (Warner Instrument Corp.) with a resistance of 4-7 megaohms. The intracellular pipette solution contained 130 mm KCl, 1 mm MgCl2, 5 mm K2ATP, 5 mm EGTA, 10 mm HEPES, adjusted with KOH to pH 7.4 (290 mOsm). The external solution contained 140 mm NaCl, 4 mm KCl, 1.8 mm CaCl2, 1.2 mm MgCl2, 11 mm glucose, 5.5 mm HEPES, adjusted with NaOH to pH 7.4 (310 mOsm). Series resistances (8-15 megaohms) were compensated (75-90%) and periodically monitored. COS7 cells were grown on 13-mm diameter coated glass coverslips in 24-well plates. Cells were rinsed for 5 min in PBS and subsequently fixed for 20 min in 4% paraformaldehyde in PBS. Following extensive washes in PBS, the cells were permeabilized by incubation with 10% normal goat serum in PBS containing 0.2% Triton X-100 for 20 min. Cells were then washed twice for 10 min each in PBS containing 1% normal goat serum. Cells were incubated at 4 °C overnight with a rabbit anti-KCNQ1 (Alomone Laboratories) channel or mouse anti-FLAG (M2; Sigma) antibodies. After a 3 × 5-min wash in PBS, cells were incubated for 1 h at room temperature with secondary antibodies, CY2-conjugated anti-rabbit IgG (1:200; Jackson Immunoresearch) and RRX-conjugated anti-mouse IgG (1:1000; Jackson Immunoresearch), respectively. Cells were viewed, and digital images were taken using a Zeiss 510 META confocal microscope using the 488-nm argon or 543-nm HeNe excitation laser lines. Biotinylation of surface proteins was carried out by incubating cells with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce) in PBS containing 1 mm phenylmethylsulfonyl fluoride and 5 mm EDTA (solution A) for 30 min at room temperature. The reaction was terminated by incubating cells for 5 min in solution A containing 20 mm glycine, followed by three washes in solution A. Cells were lysed in buffer P (50 mm Tris-HCl, pH 7.5, 20 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1.5 mm MgCl2, 10% glycerol (w/v), 1% Triton X-100 (v/v)), containing 1 mm phenylmethylsulfonyl fluoride and 10 μl/ml protease inhibitor mixture (Sigma), for 1 h at 4 °C under rotation. Cell lysate was cleared by centrifugation, and biotinylated proteins were precipitated by incubation with streptavidin-agarose beads (Pierce) overnight at 4 °C. The beads were washed six times with buffer P. Proteins were eluted by incubation with sample buffer at room temperature and then resolved by SDS-PAGE and Western blotted. Peptide samples were suspended at room temperature in a mixture of metal chloride salts containing Tris (10 mm), pH 7.5, 100 mm NaCl, and a 20 μm concentration of the indicated metals for 2 h at 0.35 mg/ml. Subsequently, peptide was dialyzed for 16 h against the same buffer without metals. Samples were prepared for analysis by incubation in a hot water bath at 80 °C for 2 h with the following addition. One-ml volumes of concentrated nitric acid were added to 1-2-ml sample batches in polypropylene 50-ml tubes. Liquid residues were taken up in deionized water and made up to a 10-ml volume with the same solvent. Metal concentration in the acid extract was determined by ICP atomic emission spectrometry. An ICP atomic emission spectrometer with cross-flow nebulizer was used (Spectroflame Modula E from Spectro, Kleve, Germany). Oligomeric State of the KCNQ1 COOH Terminus-CaM Complex—Work by Pitt and co-workers (11Ghosh S. Nunziato D.A. Pitt G.S. Circ. Res. 2006; 98: 1048-1054Crossref PubMed Scopus (136) Google Scholar) suggested previously that a soluble KCNQ1 COOH terminus-CaM complex is tetrameric using chemical cross-linking, consistent with the universal tetrameric structure of Kv channels. Using a similar molecular complex, comprising soluble COOH terminus and constitutively bound CaM (Fig. 1, B and C), we probed its oligomerization state (i.e. its molecular weight) by sedimentation equilibrium analysis. The results indicate that the complex is best modeled as a tetramer with four subunits of KCNQ1 COOH terminus and four bound molecules of CaM (Fig. 1D). Data analysis, assuming a single sedimenting solute species, yields a buoyant molecular weight of 54,000 ± 3000 (solid line). This value is very close to that calculated from the addition of the buoyant molecular weights of four molecules of the COOH terminus (4 × 8400) and four of CaM (4 × 4500). It is not compatible with a 4:2 complex (dotted line). Sedimentation velocity studies that evaluate homogeneity/dispersity of the same COOH terminus-CaM complex provide convincing evidence that the primary species (89-95% of the total loading concentration, varying by experiment) is this very same tetrameric complex (8.2 S) with a minor species that represents a higher molecular weight aggregate (11.4 S) (Fig. 1E). Importantly, there is no evidence of a free CaM species, indicating that CaM is stably bound, since measurements of CaM alone exhibit a symmetrical sharp distribution with a standard s value of 2.8 S, compatible with the expected value for monomeric CaM (Fig. 1E). No such peak is detected in the profile of the COOH terminus-CaM complex. The hydrodynamic behavior of the 4:4 COOH terminus-CaM complex (frictional ratio f/f0 = 1.8; see Ref. 29Waxman E. Laws W.R. Laue T.M. Nemerson Y. Ross J.B. Biochemistry. 1993; 32: 3005-3012Crossref PubMed Scopus (74) Google Scholar) is compatible with the solute being an elongated, nonglobular protein. Moreover, calcium is not required for this assembly, since size exclusion chromatography of a COOH terminus-CaM1234 complex, utilizing a CaM mutant that does not bind calcium, behaves like a COOH terminus-CaMWT complex (Fig. 1F). CaM Organization—To explore the possibility that CaM may induce oligomerization by its association with the COOH-terminal proximal half, we coexpressed CaM with a protein segment spanning helix A and B but truncated afterward (Δ helices C-D plus CaM) (Fig. 1, B and C). Sedimentation velocity and size exclusion chromatography indicates that it behaves as a 1:1 complex of CaM to Δ helices C-D; i.e. CaM does not cross-link COOH terminus subunits, inducing their oligomerization (Fig. 1E and supplemental Fig. 1A), neither in the presence of calcium (1 mm) nor in its absence (5 mm EDTA). Since the intervening loop between helices A and B is long, binding by CaM of these two helices may be in either parallel or anti-parallel helical arrangements. Nevertheless, the loop is not necessary for CaM binding, since its removal does not prevent formation of the Δ loop-CaM complex (Fig. 1, B and C), consistent with an anti-parallel helix bundle as the target of CaM. Role of the Distal COOH Terminus (Helices C and D)—Earlier studies on KCNQ have assigned regions from the distal COOH terminus, encompassing sequences predicted to form the two helices C and D with what has been called an assembly or tetramerization domain. Both of these predicted heli
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