Calcium, membrane fusion and exocytosis in Paramecium tetraurelia

Abstract

The involvement of plasma membrane (PM) receptor-operated calcium channels (ROCCs) was demonstrated in the exocytosis of trichocyst matrices (tmxs) from Paramecium tetraurelia. Exocytosis required external Ca{dollar}\sp{lcub}2+{rcub}{dollar}, but was independent of K{dollar}\sp{lcub}+{rcub}{dollar} channels or external Na{dollar}\sp{lcub}+{rcub}{dollar}. Exocytosis was inhibited by treatment with (i) the Ca{dollar}\sp{lcub}2+{rcub}{dollar} channel blockers verapamil and Cd{dollar}\sp{lcub}2+{rcub}{dollar} (but not nifedipine), and (ii) pronase.;Under current-clamp, exocytosis was accompanied by a transient hyperpolarization that was eliminated by K{dollar}\sp{lcub}+{rcub}{dollar} channel blockers, unmasking a transient depolarization. Under whole-cell voltage-clamp and K{dollar}\sp{lcub}+{rcub}{dollar} channel blocking conditions, secretagogue stimulated an inward current suggesting that external Ca{dollar}\sp{lcub}2+{rcub}{dollar} enters the cell through ROCCs as part of the exocytosis signal transduction pathway.;"Pseudoexocytosis" (psdx) stimulation, defined as expansion of tmxs without membrane fusion, resulted in the ejection of tmxs with patches of PM attached, as demonstrated by electron microscopy and immunoblot. Psdx was stimulated with paranitrophenol (pNP) in wild type cells, the exo-mutants nd6 and nd9, and in the ciliary Ca{dollar}\sp{lcub}2+{rcub}{dollar} channel mutants dancer and pawn. PNP-induced psdx was independent of external Ca{dollar}\sp{lcub}2+{rcub}{dollar}, and unaffected by verapamil blockade or pronase treatment, suggesting an intracellular site of action of pNP.;The PM located at the trichocyst docking site is hypothesized to contain the ROCCs, and other proteins necessary for exocytosis, including the "fusion rosette particles". In order to enrich for this membrane, tmxs released by psdx (with attached PM) were isolated on a percoll gradient. The proteins of the attached membranes were separated from the tmx proteins by extraction with detergent. SDS PAGE revealed scores of protein bands, and differences between wt and nd6 extracts.