Amperometric Measurements at Cells Support a Role for Dynamin in the Dilation of the Fusion Pore during Exocytosis
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Abstract Dynamin is a GTPase mechanochemical enzyme involved in the late steps of endocytosis, where it separates the endocytotic vesicle from the cell membrane. However, recent reports have emphasized its role in exocytosis. In this case, dynamin may contribute to the control of the exocytotic pore, thus suggesting a direct control on the efflux of neurotransmitters. Dynasore, a selective inhibitor of the GTPase activity of dynamin, was used to investigate the role of dynamin in exocytosis. Exocytosis was analyzed by amperometry, thus revealing that dynasore inhibits exocytosis in a dose‐dependent manner. Analysis of the exocytotic peaks shows that the inhibition of the GTPase activity of dynamin leads to shorter, smaller events. This observation, together with the rapid effect of dynasore, suggests that the blocking of the GTPase induces the formation of a more narrow and short‐lived fusion pore. These results suggest that the GTPase properties of dynamin are involved in the duration and kinetics of exocytotic release. Interestingly, and in strong contrast with its role in endocytosis, the mechanochemical properties of dynamin appear to contribute to the dilation and stability of the pore during exocytosis.Keywords:
Amperometry
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The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. Dynamin‐2 is a ubiquitous protein that mediates endocytosis in many epithelial cells. It is not known whether NKCC2 undergoes constitutive endocytosis and whether dynamin‐2 is involved. We hypothesized that constitutive endocytosis of NKCC2 is mediated by dynamin‐2. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. First, we studied whether dynamin‐2 is expressed in TAL. We found that dynamin‐2 is expressed in rat medullary TAL suspensions as a single band at the predicted molecular weight of 100 kDa. In control TALs we observed constitutive NKCC2 endocytosis over 30 min that averaged 10.1 ± 0.8 a.u/min. We tested whether the dynamin inhibitor (Dynasore) inhibits constitutive NKCC2 endocytosis. In the presence of Dynasore (100 μM), the rate of constitutive NKCC2 endocytosis averaged 2.8 ± 0.6 a.u/min ( n = 6, p < 0.01 vs control), a 72% inhibition. Acute (20 min) blockade of endocytosis with Dynasore increased surface NKCC2 by 66 ± 26% (p<0.05). The total pool of NKCC2 was not changed by Dynasore. We concluded that constitutive NKCC2 endocytosis is mediated by dynamin‐2. These are the first data showing NKCC2 endocytosis and a role for dynamin in TALs.
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The dynamin GTPase drives receptor‐mediated endocytosis in addition to other membrane vesicle scission events. We aim to understand how vesicle interactions of the dynamin pleckstrin homology (PH) domain contribute to the conversion of basal, auto‐inhibited dynamin tetramers to an activated, multi‐oligomeric state with a >200‐fold increased rate of GTP hydrolysis. Although dynamin self‐assembly alone has been used to explain this activation, emerging evidence suggests that dynamin may have a more complex conformational interplay between its PH and GTPase domains. Through selective mutagenesis of the dynamin PH domain based on common PH‐GTPase interaction motifs in biology, we have identified an interface of the dynamin PH domain that regulates auto‐inhibition of dynamin GTP hydrolysis rates. Specifically, we find that mutations within this interface fall into two distinct groups: a "weak" class with modestly elevated basal and lipid‐stimulated GTPase activity, as well as an "uncoupled" class whose GTPase rates are elevated ~70‐fold even in the absence of lipid. Funding provided by The Jane Coffin Childs Memorial Fund for Medical Research (JAK) and NIH R01‐GM‐078345 (MAL)
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Dynamin is a large GTPase that mediates plasma membrane fission during clathrin-mediated endocytosis. Dynamin assembles into polymers on the necks of budding membranes in cells and has been shown to undergo GTP-dependent conformational changes that lead to membrane fission in vitro. Recent efforts have shed new light on the mechanisms of dynamin-mediated fission, yet exactly how dynamin performs this function in vivo is still not fully understood. Dynamin interacts with a number of proteins during the endocytic process. These interactions are mediated by the C-terminal proline-rich domain (PRD) of dynamin binding to SH3 domain-containing proteins. Three of these dynamin-binding partners (intersectin, amphiphysin and endophilin) have been shown to play important roles in the clathrin-mediated endocytosis process. They promote dynamin-mediated plasma membrane fission by regulating three important sequential steps in the process: recruitment of dynamin to sites of endocytosis; assembly of dynamin into a functional fission complex at the necks of clathrin-coated pits (CCPs); and regulation of dynamin-stimulated GTPase activity, a key requirement for fission.
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