Analysis of TRPC3-Interacting Proteins by Tandem Mass Spectrometry

Abstract

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.

Fast endocytic recycling determines TRPC1–STIM1 clustering in ER–PM junctions and plasma membrane function of the channel

“…Conclusive evidence for TRPC1 internalization by Arf6 was shown by the reduction in regulated trafficking of TRPC1 to the plasma membrane after store depletion. This finding is different from previous findings with TRPC3 and TRPC5 that are internalized via clathrin-dependent pathway [44,45]. The variations in channel internalization could be dictated by the variations in their regulatory mechanisms, e.g.…”

TRPC3/6/7: Topical aspects of biophysics and pathophysiology

“…17 Notably, the importance of TRPC3 in neuronal growth was recently emphasized also by a proteomic study that revealed a physical link between TRPC3 and proteins involved in neuronal growth. 18 A similar conclusion was reached for TRPC6 in a study that identified TRPC6 as molecular target of hyperforin, a bicyclic polyprenylated acylphloroglucinol derivate with antidepressive effects. Hyperforin was shown to induce neuronal axonal sprouting that was strictly TRPC6 dependent.…”

The dynamic complexity of the TRPC1 channelosome

“…32,[37][38][39][40] Biochemical evidence has also been provided for the association of G proteins, SERCA and PMCA as well as PLC with TRPC channels. It is interesting to note that TRPCs have the ability to interact with both plasma membrane as well ER-associated proteins (these have also been confirmed in proteomic studies for TRPC3 3,41 ). In addition, some TRPC proteins also bind lipids such as PIP 2 and possibly cholesterol.…”