TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane

doi:10.1073/pnas.0800881105

TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane

Department of Cardiology, Howard Hughes Medical Institute, Children's Hospital Boston, and Department of Neurobiology, Harvard Medical School, Enders Building 1309, 320 Longwood Avenue, Boston, MA 02115

Contributed by David E. Clapham, January 29, 2008 (received for review December 3, 2007)

Abstract

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.

Footnotes

^†To whom correspondence should be addressed. E-mail: dclapham{at}enders.tch.harvard.edu
Author contributions: S.B. and G.K. contributed equally to this work. S.B., G.K., and D.E.C. designed research; S.B., G.K., and L.K. performed research; S.B., G.K., and L.K. contributed new reagents/analytic tools; S.B., G.K., and D.E.C. analyzed data; and S.B., G.K., and D.E.C. wrote the paper.
↵*Present address: Laboratorio de Fisiologia Sensorial, 407-D, Instituto de Fisiologia, Facultad de Medicina, Universidad Austral de Chile, Campus Isla Teja, Valdivia, 511-0566, Chile.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0800881105/DC1.
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