© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Calsequestrin mutant D307H exhibits depressed binding to its protein targets and a depressed response to calcium
Wayne State University, Elliman Building, Room 1107, 421 East Canfield Avenue, Detroit, MI 48201, United States
* Corresponding author. Tel.: +1 313 577 8734; fax: +1 313 577 8615. Email address: s.cala{at}wayne.edu
Objective: A point mutation in human cardiac calsequestrin (CSQ-D307H) is responsible for a form of polymorphic ventricular tachycardia (PVT). When overexpressed in heart cells, the mutated CSQ leads to diminished Ca2+ transients, consistent with defective regulation of intralumenal sarcoplasmic reticulum (SR) Ca2+.
Methods: To analyze the D307H mutant and determine whether the D307H mutation results in loss of normal protein–protein interactions, we prepared recombinant human wild-type (WT) and D307H forms of CSQ in mammalian cells.
Results: Although we found the two proteins to undergo similar glycosylation and phosphorylation, we discovered that Ca2+-dependent binding of the D307H mutant to both triadin-1 and junctin was reduced by greater than 50% compared to WT. Reduced binding of the D307H mutant CSQ to target proteins was similar throughout a complete range of Ca2+ concentrations. To investigate the mechanism of reduced Ca2+-dependent binding, Ca2+-dependent changes in intrinsic fluorescence emission for the two protein forms were compared. Intrinsic fluorescence of the D307H mutant was highly reduced, reflecting significant alteration in the tertiary protein structure. Moreover, the changes in fluorescence caused by increasing the Ca2+ concentration were very significantly blunted, indicating that the Ca2+-dependent conformational change was virtually lost.
Conclusions: We conclude that the point mutation D307H leads to a profoundly altered conformation that no longer responds normally to Ca2+ and fails to bind normally to triadin and junctin.
KEYWORDS Calsequestrin; D307H; Junctin; Triadin-1; Conformation; Ca2+
Time for primary review 21 days
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