Massive calcium-activated endocytosis without involvement of classical endocytic proteins

• Published in: The Journal of general physiology Vol. 137; no. 1; pp. 111 - 132
• Main Authors: Lariccia, Vincenzo, Fine, Michael, Magi, Simona, Lin, Mei-Jung, Yaradanakul, Alp, Llaguno, Marc C, Hilgemann, Donald W
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.01.2011; The Rockefeller University Press
• Subjects: Actin Cytoskeleton - drug effects; Adenosine triphosphatase; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Animals; beta-Cyclodextrins - pharmacology; Calcineurin - metabolism; Calcium; Calcium - metabolism; Calcium - pharmacology; Cell Membrane - metabolism; Cell Membrane - physiology; Cells, Cultured; Ceramides - pharmacology; Cholesterol - pharmacology; Clathrin - metabolism; Cricetinae; Cytoplasm; Cytoskeleton; Dynamins - metabolism; Electric Capacitance; Endocytosis; Endocytosis - drug effects; Endocytosis - physiology; Estrenes - pharmacology; Exocytosis - drug effects; Exocytosis - physiology; Gene expression; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; HEK293 Cells; Humans; Inositol Polyphosphate 5-Phosphatases; Kidney - drug effects; Kidney - metabolism; Lipids - physiology; Membrane Proteins - metabolism; Naphthalenes - pharmacology; Phosphoric Monoester Hydrolases - metabolism; Phosphoric Monoester Hydrolases - pharmacology; Physiology; Polyamines; Polyamines - pharmacology; Proteins; Pyridinium Compounds - analysis; Pyrones - pharmacology; Pyrrolidinones - pharmacology; Quaternary Ammonium Compounds - analysis; Sodium-Calcium Exchanger - metabolism; Sodium-Calcium Exchanger - pharmacology; Sphingomyelin Phosphodiesterase - pharmacology
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.201010468

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.