Abstract
Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiTMAB inhibited dynamin GTPase activity, with a Ki of 940 ± 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.
Footnotes
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This work was supported by grants from the National Health and Medical Research Council (Australia) and The Wellcome Trust (Ref. 062841).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.034207.
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ABBREVIATIONS: RME, receptor-mediated endocytosis; SVE, synaptic vesicle endocytosis; Tf, transferrin; EGF, epidermal growth factor; PH domain, pleckstrin homology; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; MiTMAB, myristyl trimethyl ammonium bromide; OcTMAB, octadecyl trimethyl ammonium bromide; DoTMAB, dodecyl trimethyl ammonium bromide; PS, l-phosphatidylserine; PMSF, phenylmethylsulfonyl fluoride; PFA, paraformaldehyde; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; TxR, Texas Red; A, Alexa; DAPI, 4,6-diamidino-2-phenylindole; AM, acetoxymethyl ester; DMSO, dimethyl sulfoxide; GST, glutathione transferase; PSB, phosphate-buffered saline; CI, confidence interval; HRP, horseradish peroxidase; EGFP, enhanced green fluorescent protein; pEGFP, EGFR phosphotyrosine; GFP, green fluorescent protein; PLC, phospholipase C; cmc, critical micellar concentration; WT, wild type; S, stimulation; SV, synaptic vesicle; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; FM2-10, N-(3-triethylammonium propyl)-4-(4-diethylamino)styryl)pyridinium dibromide.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Received January 17, 2007.
- Accepted August 16, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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