A ROLE FOR CALMODULIN IN THE REGULATION OF SECRETION IN PARAMECIUM TETRAURELIA

Abstract

Paramecium produce a large number of membrane-bounded secretory organelles called trichocysts. The content, the trichocyst matrix (tmx) is released following stimulation via Ca('2+)-dependent membrane fusion of trichocyst and plasma membranes.;A non-secreting, ts mutant, nd9, which lacks plasma membrane specializations required for secretion (fusion rosettes) at the non-permissive temperature recovers secretory capacity following microinjection of wt cytoplasmic factor(s). The factor is heat-labile and non-dialyzable. Preliminary studies indicate it is present in both membrane and soluble fractions which might suggest that the factor exists in both cytoplasmic and membrane-bound forms. The kinetics of recovery suggest that rosette assembly is involved. These preliminary results suggest a role for this factor in the interactions of secretory organelle and plasma membranes which lead to rosette assembly.;The Ca('2+)-dependent regulatory protein, calmodulin (CaM) is shown to be present in these cells and the role of CaM in exocytosis is investigated using CaM antagonists trifluoperazine and W-7. CaM antagonists reversibly inhibit tmx release in vivo. Ultrastructural examination of cells exposed to CaM antagonists reveals that expansion of the tmx which accompanies normal release is blocked. Inhibition is dose-dependent and varies with extracellular {lcub}Ca('2+){rcub} and drug concentration.;CaM in Paramecium is both cytoplasmic and a component of the tmx itself. In vitro, expansion of isolated, membrane free, condensed tmx is elicited by (mu)M Ca('2+), and is unaffected by CaM antagonists. Thus, inhibition of expansion in vivo is not due to a direct interaction of these agents with tmx CaM, but probably cytoplasmic CaM.;Ca('2+)-induced in vitro expansion is pH sensitive, inhibited below pH 6.6 and potentiated above pH 7.0, suggesting that factors in addition to Ca('2+) may regulate expansion in vivo. A model for tmx expansion is proposed in which pH within the trichocyst is low, favoring the condensed state. Following stimulation, cytoplasmic Ca('2+) rises, Ca('2+)-CaM complexes form, and activate Ca('2+) accumulation into the trichocyst, possibly via a proton-Ca('2+) exchange mechanism in the trichocyst membrane. Coordinated changes in Ca('2+) and pH would promote tmx expansion, and release would follow exocytic membrane fusion.