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The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.

ABSTRACT: Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses-slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic proteins. The calcium/calmodulin/calcineurin signaling pathway may underlie regulation of endocytosis by nerve activity and calcium as reported at many synapses over the last several decades.

SUBMITTER: Sun T

PROVIDER: S-EPMC2954493 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.

Sun Tao T Wu Xin-Sheng XS Xu Jianhua J McNeil Benjamin D BD Pang Zhiping P ZP Yang Wanjun W Bai Li L Qadri Syed S Molkentin Jeffery D JD Yue David T DT Wu Ling-Gang LG

The Journal of neuroscience : the official journal of the Society for Neuroscience 20100901 35

Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses-slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers ...[more]

PMID: 20810903

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Project description: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'-[?-thio]triphosphate (GTP?S). Block by GTP?S is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTP?S. 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 ?-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.

Project description:Calcium homeostasis and signaling processes in Saccharomyces cerevisiae, as well as in any eukaryotic organism, depend on various transporters and channels located on both the plasma and intracellular membranes. The activity of these proteins is regulated by a number of feedback mechanisms that act through the calmodulin-calcineurin pathway. When exposed to hypotonic shock (HTS), yeast cells respond with an increased cytosolic calcium transient, which seems to be conditioned by the opening of stretch-activated channels. To better understand the role of each channel and transporter involved in the generation and recovery of the calcium transient-and of their feedback regulations-we defined and analyzed a mathematical model of the calcium signaling response to HTS in yeast cells. The model was validated by comparing the simulation outcomes with calcium concentration variations before and during the HTS response, which were observed experimentally in both wild-type and mutant strains. Our results show that calcium normally enters the cell through the High Affinity Calcium influx System and mechanosensitive channels. The increase of the plasma membrane tension, caused by HTS, boosts the opening probability of mechanosensitive channels. This event causes a sudden calcium pulse that is rapidly dissipated by the activity of the vacuolar transporter Pmc1. According to model simulations, the role of another vacuolar transporter, Vcx1, is instead marginal, unless calcineurin is inhibited or removed. Our results also suggest that the mechanosensitive channels are subject to a calcium-dependent feedback inhibition, possibly involving calmodulin. Noteworthy, the model predictions are in accordance with literature results concerning some aspects of calcium homeostasis and signaling that were not specifically addressed within the model itself, suggesting that it actually depicts all the main cellular components and interactions that constitute the HTS calcium pathway, and thus can correctly reproduce the shaping of the calcium signature by calmodulin- and calcineurin-dependent complex regulations. The model predictions also allowed to provide an interpretation of different regulatory schemes involved in calcium handling in both wild-type and mutants yeast strains. The model could be easily extended to represent different calcium signals in other eukaryotic cells.

Dataset Information

The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.

Publications

The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.

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