7 research outputs found
Analysis of TRPC3-Interacting Proteins by Tandem Mass Spectrometry
No full textMammalian 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
Recent Progress in the Regulation of TRPC1 by Store Depletion
No full textStore-operated Ca entry (SOCE) is activated in response to depletion of the ER-Ca stores. The ER-Ca
2+
sensor protein, STIM1, oligomerizes when [Ca ] in the store is decreased and moves to ER/PM junctional
domains where it interacts with and activates channels involved in SOCE, namely Orai and TRPC channels.
2+
Orai1 is the primary pore-forming component of the highly Ca selective CRAC channel. It is recruited to ER/
PM junctional domains by STIM1 where it is gated via interaction with a specific C-terminal domain of STIM1.
Thus Orai1 and STIM1 are sufficient for generation of functional CRAC channels. Store depletion also leads
to activation of relatively non-selective cation channels, referred to as SOC channels that contribute to SOCE
in several other cell types. TRPC1 has been proposed as a possible candidate component of SOC channels.
2+
TRPC1 contributes to endogenous SOCE in many cells types. In these cells, TRPC1-mediated Ca entry and
3+
cation currents are stimulated with either agonist or thapsigargin, and inhibited by low [Gd ] and 10-20 µM
2APB (conditions that block SOCE). STIM1 also associates with and gates TRPC1 via electrostatic interaction
between STIM1 (684KK685) and TRPC1 (639DD640). Further, functional Orai1 is required for activation of
TRPC1-SOCE and this has been associated with recruitment of a TRPC1/STIM1/Orai1 complex. However,
there is ongoing debate regarding the activation of TRPC1 by store depletion as well as the role of Orai1 and
STIM1 in regulating its function. This chapter will summarize recent studies and concepts regarding the contri-
butions of Orai1 and TRPC1 to SOCE. We will discuss major unresolved questions regarding functional inter-
action between Orai1 and TRPC1 as well as possible mechanisms involved in the regulation of TRPC channels
Analysis of TRPC3-Interacting Proteins by Tandem Mass Spectrometry
No full textMammalian 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
