Calsequestrin mutant D307H exhibits depressed binding to its protein targets and a depressed response to calcium.
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 Ca(2+) transients, consistent with defective regulation of intralumenal sarcoplasmic reticulum (SR) Ca(2+).


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 Ca(2+)-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 Ca(2+) concentrations. To investigate the mechanism of reduced Ca(2+)-dependent binding, Ca(2+)-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 Ca(2+) concentration were very significantly blunted, indicating that the Ca(2+)-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 Ca(2+) and fails to bind normally to triadin and junctin.
Houle
Timothy D
TD
Wayne State University, Elliman Building, Room 1107, 421 East Canfield Avenue, Detroit, MI 48201, USA.
Ram
Michal L
ML
Cala
Steven E
SE
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002155:Calsequestrin / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017180:Tachycardia, Ventricular / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; C498269:TRDN protein, human; C070048:triadin; D006899:Mixed Function Oxygenases; C419389:ASPH protein, human; D002118:Calcium