Functional domains of sarcoplasmic reticulum Ca²⁺-ATPase studied by site-directed mutagenesis

Abstract

The availability of the cDNA clones of P-type ATPases has opened up the possibility for introducing defined point mutations in the proteins by in vitro oligonucleotide-directed mutagenesis of the cDNA. This approach can be used to search for residues involved in ligand binding or in critical conformational transitions, provided a suitable system is available for functional expression of the mutant cDNA. In addition to the requirement for a high expression level of the exogenous mutant cDNA, the absence of a significant contribution from endogenous pumps is demanded. The sarcoplasmic reticulum Ca²⁺-ATPase cDNA has been succesfully expressed in COS-1 cells, at levels more than 100-fold higher than the expression level of the endogenous COS-1 cell Ca²⁺-pump. This has permitted the functional analysis of more than 200 different Ca²⁺-ATPase mutants (2–11,16–17,19–22,24). The general strategy has been to assay first for the ATP-driven active Ca²⁺ uptake and ATPase activity in the isolated microsomal fraction containing the mutant enzyme, and thereafter for phosphorylation from ATP and P_i, its Ca²⁺ and substrate dependence, as well as the dephosphorylation kinetics in the presence and absence of ADP. Recently, it has proved possible to measure Ca²⁺-occlusion in the mutants (24).