TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane - PubMed (original) (raw)

TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane

Sebastian Brauchi et al. Proc Natl Acad Sci U S A. 2008.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Endogenous TRPM7 in SSLVs in PC12 cells. (A) Sucrose gradient distribution of the PC12 postnuclear supernatant membranes. Western blots show the association of TRPM7, synaptophysin, and VAChT (+/− pHluorin tag) with SSLV fractions. Expressed VAChT-pHluorin was detected with GFP antibody. (B) Immunofluorescent confocal image of a nontransfected PC12 cell labeled by anti-TRPM7 antibody. TRPM7 is labeled in vesicles packing the cell cytoplasm and is excluded from the nucleus. (Scale bar: 5 μm.)

Fig. 2.

Fig. 2.

Cholinergic vesicle fusion in PC12 cells monitored by VAChT-pHluorin fluorescence. (A) Single-frame TIRF image of PC12 cell expressing VAChT-pHluorin. (Insets) Selected frame sequences from fast, slow, and sustained events. (B) Example of fluorescence intensity decay time courses for transient (fast) and full-fusion (slow) events. Solid lines are the single exponential fit of the measured points. (C) Bar plot summarizes the kinetics of flash fluorescence decay in VAChT-pHluorin-expressing PC12 cells. The events segregate into fast (τ = 0.68 ± 0.28 s) and slow (τ = 3.25 ± 0.42 s) populations. Shown are 54 total events in 20 cells. (Error bars: ± SEM.)

Fig. 3.

Fig. 3.

Reduction in TRPM7 channel activity decreases SSLV fusion event frequency. (A) Population analysis of the spontaneous and stimulation-evoked SSLV fusion events with the plasma membrane. For each recorded cell, the total number of fusion events (transient, full, and sustained) in 300 frames was divided by the cell area and normalized to the average accumulated fusion number in vector-transfected cells. TRPM7 knockdown (siRNA; P < 0.05; 118 total recorded fusion events, 9 cells) and dnTRPM7 expression (dnM7; P < 0.001; 132 total recorded fusion events, 12 cells) substantially reduced fusion frequency. In contrast, overexpression of wt TRPM7 (M7-wt) and kinase-dead TRPM7 (M7-KA) did not alter fusion frequency. Fusion frequency was not significantly different in mock-transfected cells compared with nontransfected cells (data not shown). A total of 56 cells were recorded. (Error bars: ± SEM.) (B and C) Example illustrating SSLV fusions inhibition with dnTRPM7. (B) Simultaneous imaging of nontransfected and dnTRPM7-transfected cells. Image is the sum of 300 background-subtracted frames. (C) Three-dimensional projection of intensity (z; arbitrary units) and x, y pixel location of the image in A.

Fig. 4.

Fig. 4.

Increased vesicle mobility in kinase-dead TRPM7-transfected PC12 cells. (A) Color-coded mobility images reflect variation of a nonfusing vesicle's position within a 30-s recording (300 frames, see Materials and Methods). (Left) TRPM7-KA transfected cell. (Right) Mock-transfected cell. (B) Distribution histogram showing fraction of pixels with the specified mobility. Relative mobility values are defined in the Materials and Methods. Bars at top define the regions integrated. (C) Population statistics of the integrated relative pixel mobility in TRPM7-transfected PC12 cells. Pixels were separated into three groups (static: 0.01 < δ < 0.09; moderately mobile: 0.091 < δ < 0.17; and highly mobile: 0.171 < δ < 0.253). (Error bars: ± SEM.)

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