Characterization of cytoplasmic factors which complement Ca2+ channel mutations in Paramecium tetraurelia

The analysis of Ca2+-channel function in the single-celled eukaryote Paramecium can be extended to a biochemical based on recent observations that transfer of cytoplasm from wild-type cells into mutants lacking Ca2+-channel function ("pawn" mutants) causes the mutant cells to regain Ca2-channel activity. Using a convenient behavioral assay for Ca2+-channel function, we have used microinjection of cytoplasmic fractions into mutant cells to enrich for and characterize those components from wild-type cytoplasm which can "cure" cells carrying mutations in the 3 different pawn genes affecting Ca2+-channel activity (pwA,pwB, and pwC). In each case, the curing factor appears to be a protein component of an intracellular membrane. They are distinguishable on the basis of thermal, pH and divalent ion sensitivities. In addition, the factor curing the pwC mutational defect has been purified more than 180-fold. Furthermore, the pwB curing activity appears to be amplified during sequential transfer between pwB cells.