Massive calcium–activated endocytosis without involvement of classical endocytic proteins

Abstract

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 (PIP2) synthesis: PIP2 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 PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 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 PIP2 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 A2 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.

Inhibition of CaV2.3 channels by NK1 receptors is sensitive to membrane cholesterol but insensitive to caveolin-1

“…1f). Previous reports about this subject are contrasting, some agree with our observation [20,41,45], but others refer no effect on C m [43,50]. Such dissimilar results could be explained due to specific cell type and/or the particular conditions established to induce depletion of plasma membrane cholesterol (e.g., MBCD concentration, incubation time, and temperature).…”

“…In our experimental conditions, the observed decrease of C m could be explained if cholesterol depletion (1) induces endocytosis, (2) increases the thickness of plasma membrane, (3) decreases the dielectric constant value of the hydrophobic bilayer core, or (4) changes the mechanoelastic properties of the plasma membrane and/or its interaction with the cytoskeleton components. The first possibility is not supported by early studies where cholesterol depletion significantly inhibited clathrin-dependent [42] or clathrin-independent Ca 2+ -activated massive endocytosis [20]. An increased internalization of the surface membrane triggered by cholesterol depletion involving changes in membrane raft-cytoskeleton interactions, however, remains as a possibility [45].…”

Damage control: cellular mechanisms of plasma membrane repair

“…Interestingly, recent electrophysiological studies also reported massive endocytosis in cells subjected to large intracellular Ca 2+ transients, or exposed to bacterial sphingomyelinase 48, 49 . This rapid endocytic process, designated as MEND (massive endocytosis), did not involve the actin cytoskeleton or the endocytosis-regulatory proteins clathrin and dynamin 48 .…”

“…This rapid endocytic process, designated as MEND (massive endocytosis), did not involve the actin cytoskeleton or the endocytosis-regulatory proteins clathrin and dynamin 48 . These findings are similar to that observed for the sphingomyelinase-dependent endocytic process that mediates plasma membrane repair, which is also dynamin-independent 9, 16, 22 .…”

“…Experiments with inhibitors suggested that the massive Ca 2+ -dependent endocytic process designated as MEND 48, 49 might not involve the lysosomal sphingomyelinase ASM. However, it remains unclear whether full inhibition of ASM activity was achieved under those experimental conditions, and whether the large intracellular Ca 2+ transients generated by reverse Na/Ca exchange are equivalent to what occurs during physiological membrane wounding 48 .…”