TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane.
TRPM7, of the transient receptor potential (TRP) family, is both an ion channel and a kinase. Previously, we showed that TRPM7 is located in the membranes of acetylcholine (ACh)-secreting synaptic vesicles of sympathetic neurons, forms a molecular complex with proteins of the vesicular fusion machinery, and is critical for stimulated neurotransmitter release. Here, we targeted pHluorin to small synaptic-like vesicles (SSLV) in PC12 cells and demonstrate that it can serve as a single-vesicle plasma membrane fusion reporter. In PC12 cells, as in sympathetic neurons, TRPM7 is located in ACh-secreting SSLVs. TRPM7 knockdown by siRNA, or abolishing channel activity by expression of a dominant negative TRPM7 pore mutant, decreased the frequency of spontaneous and voltage-stimulated SSLV fusion events without affecting large dense core vesicle secretion. We conclude that the conductance of TRPM7 across the vesicle membrane is important in SSLV fusion.
Department of Cardiology, Howard Hughes Medical Institute, Children's Hospital Boston, MA 02115, USA.
Sebastian
S
Brauchi
Grigory
G
Krapivinsky
Luba
L
Krapivinsky
David E
DE
Clapham
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion; D016716:PC12 Cells; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D013572:Synaptic Vesicles / Q000201:enzymology / Q000502:physiology*; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*; D050494:Vesicular Acetylcholine Transport Proteins / Q000235:genetics / Q000378:metabolism
C485187:PHluorin; D034741:RNA, Small Interfering; D050053:TRPM Cation Channels; D050494:Vesicular Acetylcholine Transport Proteins; D049452:Green Fluorescent Proteins; D010770:Phosphotransferases; C486584:Trpm7 protein, rat; D000109:Acetylcholine