VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx.
The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.
Secretory Physiology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA.
Brij B
BB
Singh
Timothy P
TP
Lockwich
Bidhan C
BC
Bandyopadhyay
Xibao
X
Liu
Sunitha
S
Bollimuntha
So-Ching
SC
Brazer
Christian
C
Combs
Sunit
S
Das
A G Miriam
AG
Leenders
Zu-Hang
ZH
Sheng
Mark A
MA
Knepper
Suresh V
SV
Ambudkar
Indu S
IS
Ambudkar
D000818:Animals; D020126:Brefeldin A / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018679:Cholinergic Agonists / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050052:TRPC Cation Channels; D013744:Tetanus Toxin / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000378:metabolism
D002352:Carrier Proteins; D018679:Cholinergic Agonists; D007473:Ion Channels; D008565:Membrane Proteins; D011500:Protein Synthesis Inhibitors; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D020126:Brefeldin A; D002217:Carbachol; D002118:Calcium