calcimycin and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Multiple studies suggest that phospholipase C-gamma (PLC-gamma) contributes to regulation of sodium/hydrogen exchanger 3 (NHE3) in the small intestine, although the mechanism(s) for this regulation remain unknown. We demonstrate here that PLC-gamma binds directly to the C terminus of NHE3 and exists in similar sized multiprotein complexes as NHE3. This binding is dynamic and decreases with elevated [Ca(2+)](i). The PLC-gamma-binding site in NHE3 was identified (amino acids 586-605) and shown to be a critical regulatory domain for protein complex formation, because when it is mutated, NHE3 binding to PLC-gamma as well as NHERF2 is lost. An inhibitory peptide, which binds to the Src homology 2 domains contained in PLC-gamma without interrupting binding of PLC-gamma to NHE3, was used to probe a non-lipase-dependent role of PLC-gamma. In the presence of this peptide, carbachol-stimulated calcium inhibition of NHE3 was lost. These results mirror previous studies with the transient receptor potential channel and suggest that PLC-gamma may play a common role in regulating the cell-surface expression of ion transporters.

Topics: Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Boron Compounds; Caco-2 Cells; Calcimycin; Calcium; Carbachol; Cell Line; Humans; Immunoprecipitation; Ion Transport; Ionophores; Molecular Sequence Data; Mutation; Peptides; Phospholipase C gamma; Phosphoproteins; Protein Binding; Rabbits; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers

Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca(2+) levels (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetyloxymethyl) ester, a Ca(2+) channel blocker, Ca(2+) ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca(2+) levels, and peak Ca(2+) responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca(2+) storage compartment in cysts. The ability to sequester ions in membrane-bounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca(2+) storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence. Thus, the basal bodies may be a cellular control center to coordinate the resumption of motility and cytokinesis in excystation.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Boron Compounds; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Cell Division; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Giardia lamblia; Ionomycin; Ionophores; Kinetics; Molecular Sequence Data; Movement; Sequence Homology, Amino Acid; Signal Transduction; Thapsigargin; Time Factors; Transfection; Verapamil