Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants.
CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.
Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, OH 43210, USA. bal.8@osu.edu
Naresh C
NC
Bal
Nivedita
N
Jena
Danesh
D
Sopariwala
Tuniki
T
Balaraju
Sana
S
Shaikh
Chandralata
C
Bal
Ashoke
A
Sharon
Sandor
S
Gyorke
Muthu
M
Periasamy
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002155:Calsequestrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002412:Cations / Q000378:metabolism*; D006801:Humans; D008673:Metals, Alkaline Earth / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000032:analysis / Q000378:metabolism*; D020125:Mutation, Missense / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D055503:Protein Multimerization / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity; D017180:Tachycardia, Ventricular / Q000235:genetics* / Q000378:metabolism; D000098850:Polymorphic Catecholaminergic Ventricular Tachycardia
C443109:CASQ2 protein, human; D002155:Calsequestrin; D002412:Cations; D008673:Metals, Alkaline Earth; D050505:Mutant Proteins; D002118:Calcium