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.
Brauchi
Sebastian
S
Department of Cardiology, Howard Hughes Medical Institute, Children's Hospital Boston, MA 02115, USA.
Krapivinsky
Grigory
G
Krapivinsky
Luba
L
Clapham
David E
DE
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