Mutations in Calmodulin Cause Ventricular Tachycardia and Sudden Cardiac Death
2012; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2012.08.015
ISSN1537-6605
AutoresMette Nyegaard, Michael T. Overgaard, Mads T. Søndergaard, Marta Vranas, Elijah R. Behr, Lasse L. Hildebrandt, J. Lund, Paula L. Hedley, A. John Camm, Göran Wettrell, Inger Fosdal, Michael Christiansen, Anders D. Børglum,
Tópico(s)Cardiovascular Function and Risk Factors
ResumoCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death. Idiopathic ventricular tachycardia (VT) is a cardiac arrhythmia that is seen in individuals without structural heart disease. Depending on the ECG characteristics, it can be classified as monomorphic VT or polymorphic VT, the latter comprising a number of uncommon, often malignant familial disorders, including catecholaminergic polymorphic VT (CPVT [MIM 604772]).1Badhwar N. Scheinman M.M. Idiopathic ventricular tachycardia: Diagnosis and management.Curr. Probl. Cardiol. 2007; 32: 7-43Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar CPVT is characterized by episodic syncope and/or sudden cardiac arrest induced by exercise or acute emotion.2Coumel P. Fidelle J. Lucet V. Attuel P. Bouvrain Y. Catecholaminergic-induced severe ventricular arrhythmias with Adams-Stokes syndrome in children: Report of four cases.Br. Heart J. 1978; 40: 28-37Google Scholar, 3Napolitano C. Priori S.G. Diagnosis and treatment of catecholaminergic polymorphic ventricular tachycardia.Heart Rhythm. 2007; 4: 675-678Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar The ECG is usually within normal limits at rest and often displays prominent U waves, but ventricular arrhythmias might arise at times of adrenergic activation.4Aizawa Y. Komura S. Okada S. Chinushi M. Aizawa Y. Morita H. Ohe T. Distinct U wave changes in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT).Int. Heart J. 2006; 47: 381-389Crossref PubMed Scopus (38) Google Scholar, 5Viitasalo M. Oikarinen L. Väänänen H. Kontula K. Toivonen L. Swan H. U-waves and T-wave peak to T-wave end intervals in patients with catecholaminergic polymorphic ventricular tachycardia, effects of beta-blockers.Heart Rhythm. 2008; 5: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The arrhythmias, typically bidirectional and/or polymorphic VT, can develop into ventricular fibrillation and sudden death, leading to a high mortality rate (30%–50% by the age of 30) for this disorder.6Fisher J.D. Krikler D. Hallidie-Smith K.A. Familial polymorphic ventricular arrhythmias: A quarter century of successful medical treatment based on serial exercise-pharmacologic testing.J. Am. Coll. Cardiol. 1999; 34: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar CPVT often manifests in childhood, and a family history of juvenile sudden death and stress-induced syncope is present in approximately one-third of the cases. It can present as sudden death in children without any prior signs or warning,6Fisher J.D. Krikler D. Hallidie-Smith K.A. Familial polymorphic ventricular arrhythmias: A quarter century of successful medical treatment based on serial exercise-pharmacologic testing.J. Am. Coll. Cardiol. 1999; 34: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and it has been estimated to cause up to 15% of unexplained sudden cardiac deaths in young people.7Liu N. Ruan Y. Priori S.G. Catecholaminergic polymorphic ventricular tachycardia.Prog. Cardiovasc. Dis. 2008; 51: 23-30Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Previously, familial assessment revealed little evidence of the condition in many victims of unexplained sudden death.8Behr E.R. Dalageorgou C. Christiansen M. Syrris P. Hughes S. Tome Esteban M.T. Rowland E. Jeffery S. McKenna W.J. Sudden arrhythmic death syndrome: Familial evaluation identifies inheritable heart disease in the majority of families.Eur. Heart J. 2008; 29: 1670-1680Crossref PubMed Scopus (333) Google Scholar, 9Tester D.J. Spoon D.B. Valdivia H.H. Makielski J.C. Ackerman M.J. Targeted mutational analysis of the RyR2-encoded cardiac ryanodine receptor in sudden unexplained death: A molecular autopsy of 49 medical examiner/coroner's cases.Mayo Clin. Proc. 2004; 79: 1380-1384Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar Mutations in the ryanodine receptor 2 gene (RYR2 [MIM 180902]) are known to cause dominantly inherited CPVT10Priori S.G. Napolitano C. Tiso N. Memmi M. Vignati G. Bloise R. Sorrentino V. Danieli G.A. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia.Circulation. 2001; 103: 196-200Crossref PubMed Scopus (1145) Google Scholar (CPVT1 [MIM 604772], and more than 70 different mutations are currently known. A less common autosomal-recessive form of the disorder (CPVT2 [MIM 611938]) is caused by mutations in the calsequestrin-2 gene11Lahat H. Pras E. Olender T. Avidan N. Ben-Asher E. Man O. Levy-Nissenbaum E. Khoury A. Lorber A. Goldman B. et al.A missense mutation in a highly conserved region of CASQ2 is associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia in Bedouin families from Israel.Am. J. Hum. Genet. 2001; 69: 1378-1384Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar (CASQ2 [MIM 114251]). Mutations in these genes together explain a little more than half of all familial CPVT cases. Mutations in KCNJ2 [MIM 600681] and ANK2 [MIM 106410], normally causing Andersen-Tawil Syndrome (ATS [MIM 170390]) and type 4 long-QT syndrome (LQT4 [MIM 600919]), respectively, have in addition been found in a low number of individuals diagnosed with CPVT.12Mohler P.J. Splawski I. Napolitano C. Bottelli G. Sharpe L. Timothy K. Priori S.G. Keating M.T. Bennett V. A cardiac arrhythmia syndrome caused by loss of ankyrin-B function.Proc. Natl. Acad. Sci. USA. 2004; 101: 9137-9142Crossref PubMed Scopus (276) Google Scholar, 13Tester D.J. Arya P. Will M. Haglund C.M. Farley A.L. Makielski J.C. Ackerman M.J. Genotypic heterogeneity and phenotypic mimicry among unrelated patients referred for catecholaminergic polymorphic ventricular tachycardia genetic testing.Heart Rhythm. 2006; 3: 800-805Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Finally, a locus for a severe form of CPVT (CPVT3 [MIM 614021]) was in 2007 localized to chromosome 7 without the identification of the disease gene.14Bhuiyan Z.A. Hamdan M.A. Shamsi E.T. Postma A.V. Mannens M.M. Wilde A.A. Al-Gazali L. A novel early onset lethal form of catecholaminergic polymorphic ventricular tachycardia maps to chromosome 7p14-p22.J. Cardiovasc. Electrophysiol. 2007; 18: 1060-1066Crossref PubMed Scopus (61) Google Scholar Tightly controlled cycling of the intracellular calcium concentration is the basis for cardiac muscle contraction and determination of the heart rhythm. RYR2 is the sarcoplasmic reticulum (SR) calcium release channel. Upon a small increase in local intracellular Ca2+ concentration (from approximately 100 nM to a few micromolar), this channel switches from a closed to an open conformation, resulting in a large influx of Ca2+ from the SR storage and ultimately causing muscle contraction. The current molecular understanding of RYR2-associated VT is that RYR2 mutations render the tetrameric RYR2 complex "leaky," thereby leading to increased local Ca2+ concentrations (Ca2+ sparks), untimely activation of nearby RYR2 clusters through calcium-induced calcium release (CICR), and eventual arrhythmia.15Cerrone M. Napolitano C. Priori S.G. Catecholaminergic polymorphic ventricular tachycardia: A paradigm to understand mechanisms of arrhythmias associated to impaired Ca(2+) regulation.Heart Rhythm. 2009; 6: 1652-1659Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Kontula K. Laitinen P.J. Lehtonen A. Toivonen L. Viitasalo M. Swan H. Catecholaminergic polymorphic ventricular tachycardia: Recent mechanistic insights.Cardiovasc. Res. 2005; 67: 379-387Crossref PubMed Scopus (84) Google Scholar The precise molecular mechanism is, however, still unclear, and it has been put forward that an abnormal interaction with one or more of the associated proteins or ions, such as FKBP12.6 or CASQ2, or abnormal activation by extraluminal or intraluminal Ca2+ ions also might play a role, as reviewed by Kontula et al.16Kontula K. Laitinen P.J. Lehtonen A. Toivonen L. Viitasalo M. Swan H. Catecholaminergic polymorphic ventricular tachycardia: Recent mechanistic insights.Cardiovasc. Res. 2005; 67: 379-387Crossref PubMed Scopus (84) Google Scholar and Mohler et al.17Mohler P.J. Wehrens X.H. Mechanisms of human arrhythmia syndromes: Abnormal cardiac macromolecular interactions.Physiology (Bethesda). 2007; 22: 342-350Crossref PubMed Scopus (27) Google Scholar Here we report a Swedish multiplex family presenting with a history of ventricular arrhythmias, syncopes and sudden death, predominantly in association with physical exercise or stress (Figure 1A). The index case (II:6), a now 42-year old man of Swedish ethnic origin, presented with syncope while playing football at the age of 12. The ECG at admission (Figure 1B, upper panel) showed sinus bradycardia (HR = 45/min) and prominent U-wave in V2 and V3, but no evidence of QT prolongation (QTc = 0.42 s). He had a history of loss of consciousness on at least four occasions during physical activity and once in connection with a fire alarm. A 24 hr ECG registration revealed ventricular extrasystoles (VES), bigemini, and paired VES (Figure 1B, lower panel) during football training, but no symptoms were reported. He was started on β1-adrenergic-receptor blocker treatment. At follow up, a 24 hr ECG and exercise test still showed some VES and bigemini under conditions of increasing load and heart rate. A genomic DNA sample from the index case was later screened for mutations in a panel of arrhythmia-associated genes, including RYR2 and CASQ2, where no mutations were identified. An older brother (II:4), aged 23, had a history of repeated syncope during exercise. During an exercise test, he displayed polymorphic VT (an ECG from that time is not available). After treatment with β1-adrenergic-receptor blocker, a follow-up exercise test showed VES at high loads. The family history also included a brother (II:3) who drowned during a swimming competition at age 15 after having had prior episodes of syncope and an older sister (II:2) who suffered from fits or syncope. After a later episode of ventricular fibrillation (VF), she was stabilized by treatment with β1-adrenergic-receptor blocker and became asymptomatic. A younger sister (II:8) presented at the age of 7 with a history of repeated syncope during intense physical activity. One younger family member (III:5), with reported syncope from age 2.5, died suddenly at age 13, having been treated with β1-adrenergic-receptor blocker for several years. Another family member (III:4) started having syncope at age 4, was asymptomatic under treatment with β1-adrenergic-receptor blocker, and suffered cardiac arrest at age 16. After rapid defibrillation and resuscitation, she recovered and had an ICD implanted. An older sister (III:2) presented with syncope from age 6–7 and became asymptomatic under treatment with β1-adrenergic-receptor blocker. A younger sister (III:6) presented with syncope at age 3–4. Family members III:9, III:12, and IV:1 were put on β1-adrenergic-receptor blocker after having suffered syncope and attacks of dizziness. According to the family, subject I:1 suffered multiple cases of syncope in her youth and was on medication. She died at 60 years old. Thus, the phenotypic picture of the family is characterized by CPVT-like features with symptoms including frequent syncope and three cases of sudden death or cardiac arrest. The affected family members display no other apparent clinical manifestations. All procedures were in accordance with the ethical standards of the responsible committees (institutional and national) on human experimentation. After obtaining informed consent from all the subjects, we extracted genomic DNA from peripheral blood samples from all the subjects to perform linkage analysis. Initially, 12 subjects (nine affected and three unaffected, indicated by an asterisk in Figure 1A) were genotyped with the Human Mapping 50K SNP Xba240 Array (Affymetrix, High Wycombe, UK). The unaffected subjects were all older than 16 years at the time of inclusion. Genotypes were called by the Genotyping Console (Affymetrix) and uploaded to the BCSNP data management platform (BC Platforms, Espoo, Finland) for quality-control filtering (58,958 markers). Markers with Mendelian errors were detected with MERLIN19Abecasis G.R. Cherny S.S. Cookson W.O. Cardon L.R. Merlin—Rapid analysis of dense genetic maps using sparse gene flow trees.Nat. Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar and removed from the data set. Removal of monomorphic markers and LD pruning (for which a sliding window of 50 SNPs and an r2 threshold of 0.5 was used) was performed with PLINK20Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A. Bender D. Maller J. Sklar P. de Bakker P.I. Daly M.J. Sham P.C. PLINK: A tool set for whole-genome association and population-based linkage analyses.Am. J. Hum. Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (19629) Google Scholar and resulted in a filtered data set of 7,199 markers in approximate linkage equilibrium with each other. MERLIN was used for the identification of unlike genotypes, resulting in the removal of 292 genotypes from the data set. A parametric linkage analysis assuming autosomal-dominant inheritance with complete penetrance and a disease gene frequency of 0.0001 was carried out with MERLIN with allele frequencies from the Affymetrix 100K frequency files (CEU population) and genetic distances from the Affymetrix 100K Marshfield cM map. Two possible linked loci were identified (on chromosomes 14q31-32 and 6q27-qter), each with a maximum LOD score of 3.01 (Figure S1 in the Supplemental Data available online). A follow-up analysis with microsatellite markers (D14S1037, D14S68, D14S81, D14S65, D6S305, D6S386, and D6S1590) in these two regions was performed and included an additional six subjects from the family (one affected, IV:1; two unaffected, III:10 and III:11; and three healthy married-in individuals, II:1, II:5, and II:7). In total, 18 individuals with the microsatellite markers were genotyped. Primer sequences were retrieved from the NCBI UniSTS database. The microsatellite analysis excluded the locus on chromosome 6 and mapped the disease locus to chromosome 14 (LOD score 3.9) (Figure 2A). A haplotype analysis confirmed the presence of the chromosome 14 disease haplotype in all affected and none of the unaffected individuals (Figure 1A), suggesting 100% penetrance of the mutation in this family. The peak on chromosome 14 extended approximately 21 cM, from rs9323782 (86.6Mb) to rs721403 (95.7Mb) (hg19) (Table S2), a region that harbors around 70 genes (Figure S2 and Table S3). Because of the pivotal role of calmodulin in calcium signaling and heart contraction, CALM1 was selected for sequencing. Primers amplifying the coding regions, adjacent splice sites, and the 5′- and 3′- untranslated regions of CALM1 were designed with Primer324Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers.Methods Mol. Biol. 2000; 132: 365-386Crossref PubMed Google Scholar (Table S1). The amplified products were purified and sequenced on both strands at MWG (Eurofins MWG Operon, Ebersberg, Germany). Sequencing one affected family member (the index case) and one unaffected family member revealed a heterozygous missense mutation c.161A>T [GenBank NM_006888.4] in exon 3. This mutation results in a change from asparagine (Asn) to isoleucine (Ile), p.Asn53Ile (UniProtKB P62158, notated without the initiator methionine residue to reflect the mature processed calmodulin) in the index case (Figure 2B). A genotyping assay for p.Asn53Ile for the LightCycler 480 instrument (Roche, Hvidovre, Denmark) was developed by TIB MOLBIOL (Berlin, Germany) (Table S1). Genotyping with LightCycler 480 Genotyping Master Mix (Roche) confirmed that the p.Asn53Ile substitution was present in all affected individuals and absent in all of the unaffected individuals in the pedigree. Genotyping of 1,200 control individuals (500 Danish medical student volunteers from Aarhus University and 700 anonymous Danish blood donors) showed that the mutation was absent among these 2,400 control chromosomes. This sample size provided an 80% power to detect a variant with a minor-allele frequency of 0.001,25Collins J.S. Schwartz C.E. Detecting polymorphisms and mutations in candidate genes.Am. J. Hum. Genet. 2002; 71: 1251-1252Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar verifying that the mutation is highly unlikely to be a normal but rare sequence variation. To systematically screen for other mutations in CALM1, we developed a mutation-detection assay for each of the five coding exons on the basis of high-resolution melting (HRM) of amplified PCR products. All assays were carried out with 480 High Resolution Melting Master (Roche) and HPLC purified primers (MWG). Sixty-three individuals, referred for RYR2 mutation analysis at the Statens Serum Institute in Denmark (where genetic testing failed to identify any RYR2 mutation in 61 of these individuals), were analyzed in duplicate. All samples with aberrant melting curves, as well as ten control samples with normal melting curves, were sequenced (primer sequences are listed in Table S1). From this screening a second heterozygous CALM1 missense mutation was identified in an individual of Iraqi origin. The mutation (c.293A>G) was located in exon 5 and resulted in an asparagine-to-serine substitution (p.Asn97Ser) (Figure 2B). DNA sequencing revealed that this mutation was absent in the mother and the father, neither of whom showed signs of heart arrhythmias, demonstrating the presence of a de novo mutation in this individual. A microsatellite marker analysis confirmed the paternity relationship. The individual with the de novo CALM1 mutation was a 23-year-old female who presented at age 4 with a successfully resuscitated, out-of-hospital cardiac arrest due to VF while she was running. She made a full neurological recovery and was stabilized by treatment with β1-adrenergic-receptor blocker. An initial ECG and echocardiogram were within normal limits, and there was no evidence of QT prolongation (not shown). Evaluation of her immediate family was unremarkable. An exercise ECG and electrophysiological study undertaken on full betablockade and right ventricular and coronary angiography were within normal limits. An initial diagnosis of idiopathic VF was made at the time. Follow-up ECGs demonstrated prominent U waves in the anterior leads but no evidence of the long-QT or Brugada syndromes (Figure 1C upper panel). When she was 12 years old, further investigation including signal-averaged ECG, echocardiography, cardiac MRI, and cold pressor and procainamide tests were unremarkable. An exercise ECG while she was off β1-adrenergic-receptor blocker demonstrated ventricular ectopy with couplets and triplets of varying morphology into recovery (Figure 1C, lower panel). These appeared to be bidirectional at times. On this basis, a diagnosis of CPVT was made, but genetic testing of RYR2, CASQ2, and KCNJ2 failed to identify any mutations in these genes. In a follow-up genetic test of other arrhythmia-associated genes, including KCNQ1 (MIM 607542), KCNH2 (MIM 152427), KCNE1 (MIM 176261), KCNE2 (MIM 603796), and SCN5A (MIM 600163), no mutations were found. She fainted twice more in her teens, and at age 15 she suffered a further cardiac arrest. After recovery, an ICD was implanted, and she remained well but was diagnosed with systemic lupus erythematosus (SLE [MIM 152700]). Her mother, aged 62, was asymptomatic until developing heart failure secondary to adriamycin-induced cardiomyopathy from breast cancer treatment. She did not experience syncope or arrhythmia. An ECG demonstrated left-bundle-branch block and leftward axis deviation. Her father, aged 66, had suffered nonexertional syncope in stressful situations, consistent with vasovagal syncope. His resting ECG was normal, and exercise testing for atypical chest pain had induced ischemic changes without arrhythmia. An angiogram was reported as showing unobstructed coronaries and vasospasm. Both CALM1 mutations thus appear to induce an early-onset and highly penetrant phenotype that belongs to the severe end of the spectrum of CPVT-like arrhythmias. In accordance with this, an initial response to therapy with β1-adrenergic-receptor blocker was observed with subsequent recurrent symptoms both in the Iraqi case and in individuals III:4 and III:5 in family 1. The fact that p.Asn97Ser is a de novo substitution is fits well with the presentation of the associated disease as a highly malignant form with very early presentation and is consistent with the RYR2-associated disease, which is commonly caused by de novo mutations.26Priori S.G. Napolitano C. Memmi M. Colombi B. Drago F. Gasparini M. DeSimone L. Coltorti F. Bloise R. Keegan R. et al.Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia.Circulation. 2002; 106: 69-74Crossref PubMed Scopus (942) Google Scholar To assess whether CALM1 missense mutations exist in the general population, a systematic HRM screen of all five CALM1 coding exons were performed in 500 Danish control individuals. No missense mutations were identified among these 1,000 control chromosomes. In contrast, three rare silent polymorphisms were identified (these were present among five control individuals) (Table S4), stressing the selection pressure against missense mutations in this gene. A specific Iraqi control population was not investigated because the mutation was de novo, and the mutation rate is likely to be the same in all populations. No missense mutations in CALM1 (ENST00000356978) were found from the 1000 Genomes Project (October 2011 release) interrogated through the Project Browser #ICHG2011 (Ensembl release 63 representing an integrated set of variant calls and INDELS from low-coverage and exome sequencing data of 1,092 individuals). A literature search also failed to present evidence for any previously identified calmodulin mutations. The lack of identified missense mutations is not surprising given that calmodulin is known as one of the most conserved molecules throughout evolution, displays an identifical protein sequence in all vertebrate species, and has evolved only slightly since the divergence from plants (Figure 2C). The presence of three independent genes in the human genome (CALM1-3 [MIM 114180, 114182 and 114183]), all encoding ubiquitously expressed identical calmodulin protein molecules, further underscores the selection pressure against any amino acid changes in this classic calcium-binding protein. Calmodulin is a 148 amino acid α-helical protein containing four classical Ca2+-binding EF hands that bind one calcium ion each. It is shaped as a dumbbell and is capable of mediating a signal consisting of small intracellular Ca2+-concentration changes to a multitude of intracellular targets via fine-tuned differences in the N- and C-domain affinity for Ca2+.27Tadross M.R. Dick I.E. Yue D.T. Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.Cell. 2008; 133: 1228-1240Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar The N domain contains the lower-affinity Ca2+-binding sites I and II, and the C domain contains the high-affinity binding sites III and IV. The two identified substituted residues are located in opposite domains; the Asn53 residue is positioned on the solvent-exposed surface of the first α-helix of Ca2+-binding site II in the N domain, and the Asn97 residue is one of the Ca2+-binding residues of binding site III, located in the calmodulin C domain (Figure 2D). Calmodulin binds to and regulates the activity of a large number of intracellular proteins, but in none of the published high-resolution calmodulin structures available are the two substituted residues in direct contact with any bound protein or peptide, exemplified by the calmodulin- RYR1 peptide complex in Figure 2D. To functionally characterize the substitutions, we used a nested priming approach to amplify the CALM1 cDNA (primers are listed in Table S1) from a cDNA preparation from liver mRNA,28Overgaard M.T. Oxvig C. Christiansen M. Lawrence J.B. Conover C.A. Gleich G.J. Sottrup-Jensen L. Haaning J. Messenger ribonucleic acid levels of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein: expression in human reproductive and nonreproductive tissues.Biol. Reprod. 1999; 61: 1083-1089Crossref PubMed Scopus (96) Google Scholar and we ligated this cDNA into a modified pMAL vector (New England Biolabs, Ipswich, USA) containing a Tobacco Etch Virus (TEV) protease cleavage site. Missense mutations were introduced with QuickChange Lightning (QIAGEN) (primer sequences are listed in Table S1). The calmodulin variants were expressed in Rosetta B cells (EMD Chemicals). All buffers and reagents were prepared with purified and deionized (>18.2 Ω resistance) water and plastic vessels, and all laboratory equipment was washed in 1 M HCl and purified water so that Ca2+ contamination would be minimized. The total Ca2+ concentration of the utilized buffers was determined to be between 1 and 3 μM with the Quin-2 fluorogenic calcium indicator and Calcium Calibration Buffer Kit #1 (Invitrogen) and an Infinite M1000 fluorescence microplate reader (Tecan, Switzerland). Calmodulin proteins were purified by affinity chromatography (Amylose Resin [New England Biolabs]), TEV cleavage, anion-exchange chromatography (Source 15Q 10/10 [GE Healthcare]), and size-exclusion chromatography (Superdex 75 16/60 [GE Healthcare]). Protein concentrations were determined by absorption at 280 nm. The identity and integrity of each protein preparation was confirmed by MALDI-TOF mass spectometry (Bruker Reflex III [Bruker-Daltronics]). First, the Ca2+-binding property of native and variant calmodulin was investigated. Because calcium binding leads to a large conformational change in calmodulin and the C domain contains two tyrosine (Tyr) residues, it is possible to monitor Ca2+ binding to the C domain by measuring the changes in intrinsic Tyr fluorescence intensity.29VanScyoc W.S. Newman R.A. Sorensen B.R. Shea M.A. Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels.Biochemistry. 2006; 45: 14311-14324Crossref PubMed Scopus (12) Google Scholar Calmodulin variants (15 μM) were titrated with CaCl2 while the intrinsic Tyr fluorescence intensity at 320 nm (277 nm excitation) was recorded on a FluoroMax 4 spectrofluorometer (HORIBA Jobin Yvon) equipped with a peltier temperature controller set at 37°C. The fractional C domain Ca2+ binding demonstrated a significant reduction in the Ca2+ affinity for the p.Asn97Ser variant
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