Premium
Mutagenesis of Residues Involved in Control of the Ca 2+ Entry Pathway and Conformational Changes Associated with Ca 2+ Binding in the SR Ca 2+ ‐ATPase
Author(s) -
ANDERSEN JENS PETER,
CLAUSEN JOHANNES D.,
EINHOLM ANJA PERNILLE,
VILSEN BENTE
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb07141.x
Subject(s) - chemistry , transmembrane domain , thapsigargin , endoplasmic reticulum , transmembrane protein , mutant , crystallography , biophysics , stereochemistry , biochemistry , membrane , biology , receptor , gene
A bstract : Rapid kinetic measurements were used to study the rate of Ca 2+ dissociation from the high‐affinity Ca 2+ sites of the dephosphoenzyme (i.e., from the E 1 Ca 2 form toward the cytoplasmic side) as well as the rate of Ca 2+ binding with associated conformational changes ( E 2 → E 1 Ca 2 transition) in the wild type and mutants of the sarcoplasmic reticulum Ca 2+ ‐ATPase expressed in mammalian cells. Cluster mutations as well as single mutations in transmembrane segment M3 resulted in conspicuous effects on the rate of Ca 2+ migration. Furthermore, mutation of Asp 59 in transmembrane segment M1 to arginine exerted a profound effect on Ca 2+ interaction. The data demonstrate an important role for M3 residues in control of the Ca 2+ entry pathway and provide functional evidence in support of a close relationship between this pathway and the water‐accessible channel leading between transmembrane segments M1 and M3 in the thapsigargin stabilized E 2 structure. In addition, rapid kinetic measurements demonstrated that the hydrogen bond network involving Asp 813 of loop L6‐7 and Lys 758 of M5 is important for the E 2 → E 1 Ca 2 transition.