Accepted Manuscript Title: Trafficking mechanisms and regulation of TRPC Channels Author: Lorena Brito de Souza Indu S. Ambudkar PII: DOI: Reference:

S0143-4160(14)00081-5 http://dx.doi.org/doi:10.1016/j.ceca.2014.05.001 YCECA 1565

To appear in:

Cell Calcium

Received date: Revised date: Accepted date:

5-5-2014 15-5-2014 16-5-2014

Please cite this article as: L.B. Souza, I.S. mechanisms and regulation of TRPC Channels., http://dx.doi.org/10.1016/j.ceca.2014.05.001

Ambudkar, Trafficking Cell Calcium (2014),

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ac

ce

pt

ed

M

an

us

cr

i

*Graphical Abstract

Page 1 of 31

*Highlights (for review)

Highlights    Ca2+‐permeable TRPC channels contribute to various cellular functions 

ip t

TRPC channel trafficking is a key determinant of their plasma membrane function  This review discusses the mechanisms and pathways regulating TRPC trafficking 

Ac ce p

te

d

M

an

us

cr

The topics discussed are endocytic, recycling, as well as exocytic mechanisms 

Page 2 of 31

*Manuscript

Trafficking mechanisms and regulation of TRPC Channels. Lorena Brito de Souza and Indu S. Ambudkar

ip t

Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD

us

cr

20892.

M

an

Key words: TRPC channels, Intracellular Trafficking, Microdomains, Ca2+ signaling, Regulation

Address all correspondence to:

d

Lorena Brito de Souza, D.D.S., Ph.D. or Indu S. Ambudkar, Ph.D.

te

Molecular Physiology and Therapeutics Branch and Secretory Physiology Section

Ac ce p

NIDCR, Building 10, Room 1N-113 NIH, Bethesda MD 20892.

Phone: 301-496-5298, 301-496-1363 Email: [email protected]

1 Page 3 of 31

Abstract TRPC channels are Ca2+-permeable cation channels which are regulated downstream from receptor-coupled PIP2 hydrolysis. These channels contribute to a wide variety of cellular

ip t

functions. Loss or gain of channel function has been associated with dysfunction and aberrant

cr

physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their

us

effects on the downstream cellular processes. Such interactions also compartmentalize the Ca2+

an

signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels

M

are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in

Ac ce p

te

d

trafficking of the seven members of the TRPC family (TRPC1-TRPC7).

2 Page 4 of 31

1. Introduction Ca2+ signaling is a pivotal regulator of physiological processes that occur in various cells, which

ip t

include secretion, gene transcription, cell proliferation, and cell death. Ca2+ signaling mechanisms in cells are initiated by the generation of a Ca2+ signal, typically an increase in

cr

cytosolic [Ca2+] ([Ca2+]i), via regulation of intracellular Ca2+ release and/or Ca2+ entry mechanisms. Ca2+ pumps and transporter activities also contribute to the control of cellular Ca2+

us

homeostasis. [Ca2+]i is dynamic and tightly regulated within the physiological range of [Ca2+]i

an

required for cell function. The amplitude, spatial as well as temporal characteristics of the [Ca2+]i increase are critical as unregulated increases in [Ca2+]i are deleterious to cells. The type

M

of Ca2+ channels and the stimuli involved in their regulation vary between cells. Typically, in non-excitable cells, essential Ca2+ signals are generated in response to receptor-dependent

d

activation of phosphatidylinositol 4,5-biphosphate (PIP2)-specific phospholipase C (PLC) which

te

leads to hydrolysis of PIP2 with generation of inositol 1,4,5-triphosphate (IP3) and

Ac ce p

diacylglycerol (DAG). IP3 mediates release of Ca2+ from the endoplasmic reticulum (ER) via binding to the IP3 receptor which triggers depletion of the ER-Ca2+ store and an increase in [Ca2+]i. The [Ca2+]i increase solely due to intracellular Ca2+ release is transient in nature. More sustained increase requires the contribution of extracellular Ca2+ entry mediated by channels that are also activated in response to receptor-stimulated PIP2 hydrolysis. The two major types of Ca2+ entry mechanisms have been described; store-operated and store-independent. Storeoperated calcium entry (SOCE) has been identified as a critical Ca2+ entry pathway in nonexcitable and excitable cell functions [1, 2]. The trigger for activation of SOCE is ER-Ca2+ store depletion while refilling the store leads to inactivation of the process. Stromal interaction molecule 1 (STIM1) has been established as the main Ca2+ sensor protein in the ER that 3 Page 5 of 31

responds to store depletion, aggregates, and translocates to ER-plasma membrane (ER-PM) junctional domains where it interacts with, and activates, the channels mediating SOCE. The store-independent Ca2+ entry mechanisms are mediated by channels that directly respond to

ip t

receptor stimulation and have been suggested to be regulated by PIP2 per se or the hydrolysis

cr

product, DAG. Much less is understood regarding their mode of activation.

us

Transient Receptor Potential Canonical (TRPC) channels constitute a group of Ca2+-permeable cation channels that are activated in response to receptor-regulated PIP2 hydrolysis. Based on

an

this, the TRPC subfamily, which consists of seven members (TRPC1-TRPC7), were first proposed as molecular components of the SOCE channels. It has now been reported that only

M

some TRPC channels contribute to SOCE while others are regulated by store-independent mechanisms. TRPC channel composition differs between cells [3-5]. It is also interesting that by

d

allowing Na+ influx, some TRPCs, e.g. TRPC3 and TRPC6, can affect the membrane potential

te

of the cells and thus impact on downstream functions; e.g. in smooth muscle cells or neuronal

Ac ce p

cells [6, 7]. Notably, TRPCs do not mediate SOCE in T lymphocytes and mast cells which exhibit an inwardly rectifying, highly Ca2+-selective, current called ICRAC (Ca2+-release activated Ca2+ current) mediated by CRAC channels [8-10]. The long search for identification of CRACchannel component led to the discovery of the Orai family of Ca2+ channels. In particular, Orai1 has been established as the major pore-forming component of CRAC channels which is activated by a specific C-terminal domain in STIM1. However, alternative mechanisms have also been reported where Orai1 and Orai3 function in a STIM1-dependent, but storeindependent manner, contributing to Ca2+ entry that is regulated by arachidonic acid or leukotriene [11-13]. In contrast to Orai1, TRPC channels generate relatively linear, nonselective, cation currents termed ISOC (Store-Operated Ca2+ current), in response to agonist 4 Page 6 of 31

stimulation [4, 14]. TRPC1 was the first mammalian TRPC channel to be cloned [15, 16]. With some exceptions, studies with endogenous TRPC1 have been the most consistent in demonstrating a role for the channel in SOCE. [17, 18]. Importantly, several studies

established that STIM1 gates TRPC1 via interaction of its terminal

684KK685

residues with

in TRPC1-C-terminus and that Orai1-dependent Ca2+ entry triggers plasma membrane

cr

639DD640

ip t

demonstrated that activation of TRPC1 is determined by STIM1 and Orai1. It has now been

us

recruitment of TRPC1 (this will be discussed in the latter sections of this review). One problem in studying TRPC1 channel function is that heterologous expression of the channel does not

an

always result in increased SOCE. Part of this can be explained by the predominant localization of the endogenous and heterologously expressed channel in intracellular compartments, the

M

nature of which have not yet been clearly described, with little expression in the plasma

d

membrane. It is also possible that overexpressed channels do not assemble with the proper

te

stoichiometry with accessory proteins that might be needed to regulate their function. In contrast some other TRPC channels, e.g. TRPC3 and TRPC6, are predominantly localized in the plasma

Ac ce p

membrane. Several studies have demonstrated that TRPC1, like other TRPC channels, undergoes trafficking to the plasma membrane which is critical for the assembly of a functional channel.

TRPC channels interact with accessory proteins, including those involved in trafficking, scaffolding and regulatory proteins. It has been suggested that such interactions might determine their localization, as well as their function, within specialized plasma membrane microdomains [3]. TRPC channels have various conserved protein-interaction motifs within the N- and Cterminal regions, including those that interact with PDZ-domains, WW-repeat domains, Cav1scaffolding domains, and even lipids such as PIP2. In addition, they have ankyrin repeats and 5 Page 7 of 31

several coiled-coiled regions that appear to be very important in the assembly of the channels as well as their plasma membrane localization and possibly interaction with the cytoskeletal components. For example, expression of the N-terminus of TRPC1 leads to disruption of

ip t

TRPC1-containing TRPC channels which suggests that the channels interact via their Nterminal domains and that either the multimerization per se or sequences within the N-terminus

cr

are critical for channel trafficking to, and/or its localization in, the surface membrane [19]. It is

us

also important to note that while some TRPCs, like TRPC1, are regulated via STIM1 and storedepletion, others like TRPC6 are regulated by metabolites generated due to PIP 2 hydrolysis,

an

probably by DAG. PIP2 itself might also serve a role in the regulation of the activity as well as trafficking of the channels. Several studies now demonstrate that stimulation of cells leads to

M

regulation of the plasma membrane expression of TRPC channels which is coupled to enhanced

d

Ca2+ influx [20-24]. This can occur on a relatively slow-time scale via increased synthesis of the

te

channel and their regulators, or via an acute mode through modulation of constitutive and regulated vesicular trafficking mechanisms. Surface expression of TRPC channels can be

Ac ce p

determined by several different processes: recruitment of channels to the plasma membrane, increased exocytosis and recycling, retention of channels in the membrane via interaction with scaffolding proteins, or decreased internalization of channels. Regulation of any one of these can provide an efficient and fast control of the surface expression of the channels and thus determine their function. All these processes have been described for various TRPC channels and these will be discussed in the following sections of this review.

2. Trafficking mechanisms that can determine TRPC channel function

6 Page 8 of 31

Based on the present available data, it appears that regardless of their mode of activation, the function of TRPC channels depends on trafficking mechanisms as well as their insertion into specific microdomains in the plasma membrane, where they can be regulated by upstream

ip t

signaling mechanisms and sequentially regulate downstream cell functions. As with other ion channels and plasma membrane proteins, cells can utilize different trafficking pathways/modes

cr

for translocating TRPC channels to the plasma membrane as well as for their internalization

us

(Figure1). The biosynthetic-secretory pathway modifies the proteins at various steps beginning with their synthesis in the ER, trafficking through the Golgi apparatus, and packaging into

an

transport vesicles that carry the channel to the plasma membrane. The final step involves exocytosis which regulates the fusion of the vesicles and insertion of the channel into the plasma

M

membrane. The endocytic pathway is also critically regulated. Several different types of

d

endocytic mechanisms have been identified which are mediated by specific proteins, e.g.

te

clathrin, ADP-ribosylation factor 6 (Arf6), and caveolin. The endocytic machinery sequesters the channels and internalizes them into the endosomal compartment from where they can be

Ac ce p

delivered either to lysosomes for degradation or recycled back to the plasma membrane through recycling vesicles [25, 26]. The recycling pathway is pivotal for returning TRPC channels to the cellular surface via the fast recycling pathway that mediates trafficking of the channels directly from the early endosomes to the plasma membrane. Thus, channels that are internalized and sorted into the early endosomes can be rapidly recycled back to the plasma membrane. On the other hand, the slow recycling pathway mediates trafficking of the channel from the early endosomes to a recycling compartment and then to the plasma membrane. Another possible recycling pathway is the retrograde pathway in which the channels could be first transported into the Trans-Golgi Network (TGN) and then sorted into the secretory pathway [25, 27, 28].

7 Page 9 of 31

Vesicular transport of the channels occurs in a precise way and involves specific and selective vesicles that facilitate recognition, delivery, and fusion of the channel with the correct target membrane and cell location. Further, the vesicle associated docking and sensor proteins can

ip t

determine where and how fast the channel containing vesicles fuse with the plasma membrane. Regulated exocytosis is triggered by a stimulus and depending on the fusion mechanism

cr

involved, can be relatively fast or slow. In some cases, when the stimulus is removed, the

us

vesicles are internalized. For example, if Ca2+ is the trigger for vesicle fusion, Ca2+ sensors with specific affinities and/or rates of response control the final step of membrane fusion. Thus,

an

intracellular trafficking mechanisms impact both the spatial and temporal regulation of channel activity. Two major classes of proteins are involved in intracellular vesicle trafficking; soluble

M

N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins and Rabs. SNAREs

d

are transmembrane proteins that function in a complementary manner in which vesicle

te

membrane SNARE (v-SNARE) interacts with the target membrane SNARE (t-SNARE) leading to the formation of a trans-SNARE complex that brings the opposing membranes together to

Ac ce p

facilitate fusion [29]. These could mediate fusion within various intracellular vesicular compartments or between intracellular vesicles and the plasma membrane. Rab proteins also contribute to the specificity of vesicular trafficking. Rab GTPases constitutes the largest family of small GTPases, which switches between two conformation states; “on mode” that is GTPbound and “off mode”, that is GDP-bound. Specific Rabs are associated with every step in the vesicle transport process. Such specificity determines the type of intracellular membrane compartment through which the protein needs to be routed. These proteins also control vesicle budding, motility, uncoating (in the case of clathrin vesicles), and fusion through interactions with coat proteins, other plasma membrane components, motors and SNAREs [30].

8 Page 10 of 31

The trafficking of TRPC channels through successive membrane compartments demands a coordinated progression of membrane identities that is controlled by Rab proteins and their effectors. For example, after internalization the routing of TRPC channels into the early

ip t

endosome is regulated by Rab5 and its effectors such as early endosome antigen 1 (EEA1) and Rabenosyn-5. The lumen of early endosome is weakly acidic with pH between 5.9-6.8 and

cr

[Ca2+] ranging in 4-40μM [31, 32]. This compartment acts as main sorting station from where

us

the channels can be directly recycled back to the plasma membrane via Rab4- or Rab35containing vesicles through the fast recycling pathway or first transported to the recycling

an

compartment from where they are translocated to the plasma membrane by Rab11-, Rab15-, Rab22-, or Rab25-dependent recycling vesicles via the slow recycling pathway [28, 30, 33].

M

Alternatively, the channels can also be routed from the early endosome to the late endosome and

d

this step is regulated by Rab7 and Rab9. The luminal pH in these vesicles ranges between 6.0-

te

4.9 [34, 35]. From the late endosomes, channels can reach TGN where they either sort into the secretory pathway or can be targeted to the lysosomes for degradation. Rab9 and its effector p40

Ac ce p

regulate trafficking from late endosomes to the TGN [33]. However, if the TRPC channel is targeted for degradation, it is trafficked from the late endosomes to the lysosomes and this is regulated by Rab7, Rab24 and Rab27. The lysosomes have an acid luminal pH ranging between 4.5-5.5 and [Ca2+] around 500μM [32, 33, 36]. The surface expression and retention of the channels in the plasma membrane can be critically regulated via interaction with scaffolding proteins that are either located in the plasma membrane, just under the membrane or are recruited to the region when the channels need to be immobilized [37]. Studies have shown that Homer1 and other PDZ domain proteins such as NHERF, as well as caveolin-1 (Cav-1) interact with TRPC channels and not only regulate their 9 Page 11 of 31

plasma membrane expression but also their activation and effects on downstream targets within the cells [19, 38-40]. Thus, the physiological function of TRPC channels is dependent on intracellular trafficking mechanisms that mediate proper channel trafficking to specific

ip t

functional microdomains on the surface of the cells, components that retain the channel in the surface membrane, as well as those that mediate their internalization. Mislocalization of the

cr

TRPC channels can adversely impact their function and consequently downstream Ca2+

us

signaling events that are dependent on these channels. Some aberrant TRPC channel function and trafficking have been associated with disease and dysfunction. Below we have discussed the

an

current state of our knowledge about the trafficking mechanisms that determine the plasma

d

M

membrane localization and function of various TRPC channels.

Ac ce p

3.1. TRPC1

te

3. Mechanisms and components of TRPC channel trafficking

TRPC1 channels interact with several proteins that regulate vesicle trafficking, membrane fusion, as well as cytoskeletal rearrangement (Table 1). Interestingly, TRPC1 function is also determined by plasma membrane lipids. It has been demonstrated that plasma membrane localization and activation of TRPC1 is dependent on the integrity of lipid raft domains in the plasma membrane. Lipid raft domains are biochemically distinct cholesterol-rich regions in the plasma membrane that are also enriched in PIP2, sphingolipids (such as sphingomyelin) and gangliosides. These domains have been proposed to act as platforms for organization of protein complexes and several signaling complexes have been reported to be associated with and be regulated within lipid rafts. The cholesterol-binding protein Cav-1 is present in specific lipid raft 10 Page 12 of 31

domains called caveolar lipid rafts. These domains can be morphologically distinct; e.g. caveolae, which typically contains a large number of Cav-1-multimers, are flask-shaped compared to planar caveolar lipid raft domains, which have fewer Cav-1 proteins. Other

ip t

components such as cavin might also contribute to the morphological and structural aspects of these domains [41, 42]. Several proteins that are localized within caveolar lipid raft domains

cr

have conserved Cav-1-binding domains. Mutation of these domains typically mislocalizes these

us

proteins [19]. In addition, non-caveolar lipid raft domains that can serve as platforms for signaling complexes. Typically proteins that are acylated tend to interact with these domains.

an

further, proteins that have binding sites for negatively charged lipids also appear to partition into

M

these membrane domains.

Biochemical studies demonstrate that TRPC1 partitions into lipid rafts in resting cells which is

d

further increased following stimulation of the cells [43]. Conversely, disruption of lipid rafts

te

with cholesterol depleting agents, like methyl-β-cyclodextrin (MβCD), impairs association of

Ac ce p

TRPC1 with these domains and also disrupts SOCE in salivary gland cells [44], vascular smooth muscle cells [45], and other cell types [46-48]. TRPC1 also has Cav-1-binding sites in its N- and C-terminal domains. The N-terminal Cav-1-binding site has been shown to be involved in scaffolding the protein and localizing it to the plasma membrane. The C-terminal domains might regulate the function and or inactivation of the channel. Both plasma membrane localization and TRPC1 activation are disrupted when Cav-1 mutant is expressed in cells, Cav-1-binding domain is mutated in TRPC1, or when Cav-1 is silenced [19, 40, 44, 49-51]. These data suggested that Cav-1 serves a scaffolding role in the plasma membrane localization of TRPC1and probably determines the association of TRPC1 with lipid raft domains. Importantly, scaffolding of TRPC1 to Cav-1 is required for its regulation and interaction with Orai1 and STIM1 [19, 40, 44, 11 Page 13 of 31

52, 53]. According to the currently proposed model, in resting cells, constitutive trafficking mechanisms target TRPC1 close to the plasma membrane where it interacts with Cav-1. Upon stimulation with physiological agonists that lead to ER-Ca2+ depletion, STIM1 is translocated to

ip t

the plasma membrane and activates Orai1. Ca2+ entry via Orai1 triggers recruitment of TRPC1 into the plasma membrane. It has also been shown that under these conditions, TRPC1

cr

dissociates from Cav-1 and when SOCE is inactivated, TRPC1 dissociates from STIM1 and re-

us

associates with Cav-1. Dissociation of TRPC1 from Cav-1 is coupled to its activation by STIM1 since C-terminal 684KK685 residues of STIM1 are required both for gating TRPC1 and releasing

an

the channel from Cav-1 [40, 54]. Importantly, STIM1 also associates with lipid raft domains following store depletion. This likely involves the C-terminal lysine-rich region of STIM1

M

which is predicted to have a PIP2-binding sequence. Deletion of this region prevents the protein

d

from forming stable puncta in the periphery of the cell. Another important finding is that

te

TRPC1-STIM1 interactions are mediated within lipid raft domains and disrupted following cholesterol depletion [55]. A critical step in TRPC1 activation is its translocation to the plasma

Ac ce p

membrane in response to store-depletion. The increase in surface expression of the channel is reduced by removal of extracellular Ca2+, knockdown of Orai1, or overexpression of Orai1 dominant negative mutant [20]. Thus, activation of TRPC1 by STIM1 is critically dependent on the recruitment of TRPC1 to the plasma membrane via a mechanism that is regulated by Orai1mediated Ca2+ entry. While it is reasonable to suppose that some Ca2+ sensor protein must be involved in this process, such a protein has not yet been identified. Furthermore, the exocytic vesicles containing TRPC1 have also not yet been identified although it has been suggested that they are likely to be localized in the vicinity of the Orai1 channels in order to sense the Ca2+ entering via that channel. TIRF measurements demonstrate clustering and increase in TRPC1-

12 Page 14 of 31

containing vesicles in the sub-plasma membrane region following stimulation. Further, TRPC1 co-localizes with STIM1 and Orai1 in this region of the cells following store-depletion and biochemical evidence has been provided to show recruitment of a TRPC1/STIM1/Orai1

ip t

complex. This association not only determines the insertion of TRPC1 into the plasma membrane, but also retention of active TRPC1 channels in the plasma membrane [40, 56].

cr

Further studies will be required to determine the exact molecular components and steps involved

us

in TRPC1 trafficking and plasma membrane insertion.

an

Physiological relevance of caveolin in TRPC1 function has been reported using caveolindeficient (Cav1-/-) mice. Endothelial cells from Cav1-/- mice showed a reduction is SOCE and

M

mislocalization of TRPC1 and TRPC4 [57]. Further, loss of Cav-1 in these mice also impaired TRPC1 localization and channel function in salivary gland cells which resulted in a dramatic

d

reduction in fluid secretion [49]. Based on studies reported thus far, it is reasonable to propose

te

that interaction with Cav-1 leads to retention of TRPC1 within specific microdomains in the

Ac ce p

cells where it can be accessed by STIM1 following stimulation of cells. Further, Orai1 is also recruited in close proximity to these microdomains such that Ca2+ entry via the channel can trigger TRPC1 recruitment [58, 59]. Such dynamic regulation of TRPC1 channel expression in the plasma membrane as well as its function provides a way to rapidly increase the [Ca2+]i and Ca2+ signaling within this microdomain. More importantly, Ca2+ entry via TRPC1 is utilized by the cells for functions that are quite distinct from those regulated by Ca2+ entry via Orai1. This suggests that specific downstream targets are likely to be scaffolded very close to the channels in order to sense the Ca2+ entering via their respective pores. Homer1 also regulates TRPC1 channels. It is reported to interact with TRPC1-C-terminus (aa 644-650) and with IP3R and following cell stimulation, the TRPC1/Homer1/IP3R complex disassembles, resulting in channel 13 Page 15 of 31

activation [38]. Homer1 deficient mice (Homer1-/-) show defective calcium signaling in skeletal muscle cells due to the lack of regulation of TRPC1 by Homer1 which results in skeletal myopathy [60]. As has been described, a common feature in TRPC channels is their assembly

ip t

into protein signaling complexes that are dynamically regulated and remodeled upon stimulation. Assembly of channels into complexes allows for specificity in the interactions

cr

between them and regulatory proteins and also increases the rate of interaction. Furthermore,

us

scaffolding of such ion channel complex in specific cellular locations is critical for the

an

compartmentalization of Ca2+ signals.

TRPC1 trafficking is also dependent on proteins that are associated with cytoskeleton and

M

microtubules [61, 62]. In endothelial cells stimulated with thrombin, RhoA (small GTPase that regulates actin cytoskeleton) interacts with TRPC1 and IP3R resulting in SOCE. Further,

d

inactivation of RhoA with C3 tranferase protein or overexpression of RhoA dominant mutant

te

affects actin polymerization, assembly and surface expression of TRPC1/IP3R complex, and,

Ac ce p

consequently, decreases SOCE [61]. β-tubulin also interacts with and regulates the trafficking and function of TRPC1. In retinal epithelial cells, colchicine-induced disruption of tubulin reduces surface expression of TRPC1 and SOCE [62]. The CaM-binding protein enkurin interacts with TRPC1 and TRPC5 and can tether other proteins to this channel complex in sperm [63]. Further studies are required to identify additional molecules and mechanisms that regulate the intracellular trafficking of TRPC1 and its downstream cell functions.

3.2. TRPC2

14 Page 16 of 31

TRPC2 is a relatively poorly studied channel since it is not expressed in human cells. The channel is reported to be activated in murine sensory neurons of the vomeronasal organ (VNO) [64, 65] and in murine sperm cells [66]. The trafficking mechanism of TRPC2 is not well

ip t

understood. The channel interacts with enkurin [63] and Homer1 [38] (Table 1). In HEK-293 cells, the chaperone receptor transporting protein 1 (RTP1) regulates the translocation and

cr

activity of TRPC2. In cells cotransfected with RTP1, TRPC2 surface expression and function

us

are increased [67]. It is presently unclear how TRPC2 translocates to the cell surface in response

an

to stimulation.

M

3.3. TRPC3

d

Unlike TRPC1, TRPC3 displays a predominantly plasma membrane localization consistent with

te

the suggestions that it can be locally regulated by DAG released locally following PIP2

Ac ce p

hydrolysis [68, 69]. Studies have also shown that TRPC3 can form a heteromeric channel with TRPC1 and be activated when TRPC1 binds to and is gated by STIM1. This interaction with TRPC1 confers store-dependent regulation on TRPC3 channels [70, 71]. However, the channel also exhibits constitutive activity, especially when expressed at relatively high levels. Increased store-independent activity of TRPC3 following stimulation with agonists, such as carbachol, and growth factors, such as epidermal growth factor (EGF) and brain-derived neurotrophic factor (BDNF) have been associated with regulated trafficking of the channel to the plasma membrane and increased surface expression [21]. The trafficking is dependent on interactions of the channel with Cav-1 [72], Homer1 [39], PLCγ [73, 74], VAMP2 [69] and RFN24 [75] (Table 1).

15 Page 17 of 31

TRPC3 is also present in caveolar Ca2+ signaling microdomains where it assembles with Cav-1, IP3R, SERCA, Gq/11, PLC and ezrin in a protein complex. A study demonstrated that Cav-1 is required for the assembly of TRPC3 with IP3R, activation of cation current (Icat) and

ip t

vasoconstriction in arterial smooth muscle cells [72]. This TRPC3 signaling complex is also

cr

regulated by cytoskeleton status. Enhancement and stabilization of the cortical actin layer due jasplakinolide or calyculin A treatments promote internalization of TRPC3 signaling complex

us

and reduction of TRPC3 activity [76]. Additionally, Homer1 stabilizes the interaction between TRPC3 and IP3R, regulating the rate of TRPC3 translocation to the plasma membrane [39, 77].

an

PLC and its product PIP2 interact and regulate the anchoring of TRPC3 within the plasma

M

membrane, promoting channel retention and surface expression [73, 74]. VAMP2 controls the fusion of TRPC3-containing vesicles to the plasma membrane. Treatment with tetanus toxin

d

(TeNT), which inhibits VAMP2 activity, impairs the translocation of TRPC3 to the plasma

te

membrane upon carbachol stimulation [69]. It is shown that constitutive trafficking of TRPC3 differs from regulated trafficking in the requirement of [Ca2+]i [69]. Further studies identified

Ac ce p

other proteins that interact with TRPC3 and, possibly, regulate trafficking and activity of the channels, such as clathrin, dynamin, AP-2, syntaxin, synaptotagmin-1 [78], MxA [79] and RACK1 [68]. Additional studies should be performed to discover the molecular mechanisms that determine the trafficking and activity of TRPC3 channels and what underlies the observed differences and variations in these mechanisms.

3.4. TRPC4

16 Page 18 of 31

TRPC4 channels, like other TRPCs, also assemble with trafficking and scaffolding proteins (Table 1). Na+/H+ exchanger regulatory factor (NHERF) and ZO1 interact with TRPC4 by their PDZ domain located in the C-terminus [80, 81]. NHERF in conjunction with tyrosine kinases

ip t

regulates TRPC4 expression in the plasma membrane and its activation. Following EGF stimulation, tyrosine kinase Fyn phosphorylates TRPC4, which augments its interaction with

cr

NHERF and surface expression [22]. It is suggested that Cav-1 acts as a scaffold protein to

us

mediate assembly of TRPC4-TRPC1-IP3R complex. Knockdown of Cav-1 reduces the interaction between TRPC1 and TRPC4 with IP3R, decreasing the expression of these channels

an

in the plasma membrane and the Ca2+ influx in endothelial cells [57]. TRPC4 also interacts with

te

3.5. TRPC5

d

M

Homer1 [38] and MxA [79].

Ac ce p

TRPC5 channels interact with dynamin, clathrin, AP-2 [82], MxA [79] and Homer [38] (Table 1). In hippocampal cells, translocation of TRPC5 to the growth cone is dependent of the interaction between TRPC5 with stathmin, a exocyst component protein [83]. TRPC5 channels are localized in intracellular vesicles close to the plasma membrane in neuronal cells. Following stimulation with EGF and Nerve Growth Factor (NGF), TRPC5-containing vesicles are rapidly translocated and inserted into the plasma membrane, increasing the surface expression and activity current of the channel. It was shown that phosphatidylinositide 3-kinase (PI(3)K), Rho, Rac1, and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)K) regulate TRPC5 intracellular trafficking and surface expression. Notably, Rac1 is required for the surface expression of homomeric TRPC5 channels but not for heteromeric TRPC1/TRPC5 channels. One possible 17 Page 19 of 31

explanation is because homomeric channels are localized in growth cones and modulate elongation, while heteromeric channels are localized in the neurites [84]. In kidney podocytes, TRPC5 and Rac1 are present in the same molecular complex. Ca2+ influx through TRPC5

ip t

activates Rac1 promoting a loss of actin stress fibers that results in increased motility of the cells [23]. Studies are required to elucidate how others proteins interact with TRPC5 and control

us

cr

the trafficking and activation of homomeric and heteromeric TRPC5 channels complexes.

an

3.6. TRPC6

M

Previous studies have shown the interactions between TRPC6 with MxA [79], RhoA [23], syntaxin [85], clathrin and dynamin [82] (Table 1). In HEK-293 cells stably transfected with

d

TRPC6, stimulation of muscarinic agonists triggers TRPC6 insertion into the plasma membrane,

te

which is retained there as long as the stimulus is present [24]. The GTPases Rab9 and Rab11

Ac ce p

regulate the intracellular trafficking of TRPC6 in Hela cells. Rab9 participates in the trafficking of proteins to the late endosomes and TGN. TRPC6 interacts and co-localizes with Rab9 in vesicular structures. Co-expression of the dominant-mutant of Rab9 results an augmentation of TRPC6 in the plasma membrane and consequently increased Ca2+ influx upon carbachol stimulation. Overexpression of Rab11, which is involved in recycling endosomes, also increases channel expression at the cell surface as well as Ca2+ entry. These results suggest that the intracellular trafficking of TRPC6 is regulated through the slow recycling pathway [86]. Additionally, PI(3)K kinase and its antagonistic phosphatase PTEN control the trafficking and activation of TRPC6 channels. The inhibition of PI(3)K by treatment with PIK-93, LY294002 or wortmannin reduces TRPC6 translocation to the plasma membrane and consequently Ca2+ 18 Page 20 of 31

entry in T6.11 cells. On the other hand, knockdown of PTEN does not affect the TRPC6 levels

ip t

in the cell surface but increases activity of the channel [87].

cr

3.7. TRPC7

Currently, there is a lack in information about trafficking of TRPC7. Only one study

an

us

demonstrates that similarly to others TRPC channels, TRPC7 also interacts with MxA [79].

M

4. Conclusion

The TRP canonical (TRPC) subfamily, which consists of seven members (TRPC1-TRPC7), are

d

Ca2+-permeable cation channels that are activated in response to receptor-mediated PIP2

te

hydrolysis via store-dependent and store-independent mechanisms. These channels are involved

Ac ce p

in a variety of physiological functions in different cell types and tissues. Of these, TRPC6 has been linked to a chanelopathy resulting in human disease. Two key players of the storedependent regulatory pathway, STIM1 and Orai1, interact with some TRPC channels to gate and regulate channel activity. The Ca2+ influx mediated by TRPC channels generate distinct intracellular Ca2+ signals that regulate downstream signaling events and consequent by cell functions. This requires localization of TRPC channels in specific plasma membrane microdomains and precise regulation of channel function which is coordinately by various scaffolding, trafficking, and regulatory proteins. Intracellular trafficking mechanisms provide a highly efficient system to deliver TRPC channels to specific sites in the cell when there is demand for their function. Indeed data are emerging to demonstrate that insertion of TRPC 19 Page 21 of 31

channels into the surface membrane can either initiate [Ca2+]i signals or modify the amplitude, frequency of oscillations, or spatial patterning of [Ca2+]i, both of which directly impact cell functions. Thus TRPC channel trafficking is an extremely critical mechanism by which cells can

ip t

achieve rapid changes in both global as well as local [Ca2+]i. Thus, it can be suggested that disruptions in trafficking of these channels would have serious consequences on cell function.

cr

As in the case of other ion channels, TRPC channels are considered potentially important targets

us

in the development of novel therapies for vascular, neuronal, and cardiac diseases. While search is ongoing for direct activators or inhibitors of the channels, modulators of trafficking

an

mechanisms can also be potentially important in altering TRPC channel function. Further studies are required to resolve between constitutive and regulated trafficking of TRPC channels

M

and determine how the two pathways contribute to [Ca2+]i signals and regulation of cell

Ac ce p

te

d

function.

20 Page 22 of 31

Table 1. Trafficking and Scaffolding proteins that interact with TRPC channels Trafficking and Scaffolding proteins

TRPC1

-tubulin [62] Cav-1 [44] Enkurin [63] Homer [38] MxA [79] RhoA [61] SNAP-25, VAMP [88] Enkurin [63] Homer-1 [38] RTP1 [67] AP-2, Clathrin, Dynamin, Synaptogamin [78] Cav-1, Ezrin [76] Homer [39] MxA [79] RACK [68] SNARES, Syntaxin, VAMP2 [69] Cav-1 [57] Homer [38] MxA [79] NHERF [81] ZOI [89] AP-2, Clathrin, Dynamin [82] EB50, NHERF [81] Enkurin[63] Homer [38] MxA [79] PI(3)K, PIP(5)K, Rac1 [84] Stathmin [83] Clathrin, Dynamin [82] MxA [79], PI(3)K, PTEN [87] Rab9, Rab11 [86] RhoA [23] Syntaxin [85] MxA [79]

cr

ip t

Channel

us

TRPC2

M

an

TRPC3

Ac ce p

TRPC5

te

d

TRPC4

TRPC6

TRPC7

21 Page 23 of 31

Figure Legends Figure1. Possible trafficking pathways of TRPC channels. Intravesicular trafficking pathways indicated in the figure regulate the localization and function of TRPC channels. The biosynthetic-

ip t

secretory pathway starts with the synthesis of the TRPC channels in the ER, passing through the Golgi apparatus and the channel delivery to the plasma membrane. At the plasma membrane, the

cr

channels are internalized by endocytosis and can be recycled back to the plasma membrane from

us

early endosomes using the fast recycling, from recycling endosomes using the slow recycling, or from the TGN by retrograde pathway. The TRPC channels can also be targeted to lysosomes for

an

degradation.

M

Figure2. Trafficking of TRPC channels to the plasma membrane. TRPC channels interact with accessory proteins, including trafficking proteins to determine their activation function and

d

localization within determined plasma membrane microdomains. In resting cells, TRPC1 is

te

present in vesicles close to the lipid rafts in the plasma membrane and interacts with Cav-1. After

Ac ce p

ER-Ca2+ depletion, STIM1 is translocated to the plasma membrane and activates Orai1. Ca 2+ entry via Orai1 recruits TRPC1 into the plasma membrane. TRPC1 dissociates from Cav-1 and is activated by STIM1. TRPC3 also assembles with Cav-1 in specific signaling microdomains rich in PIP2. VAMP2 and syntaxin regulates the fusion of TRPC3 containing- vesicles in the plasma membrane. TRPC6 trafficking is regulated by Rab9 and Rab11 vesicles, suggesting that the channel is recycled back to the plasma membrane through the slow recycling pathway.

22 Page 24 of 31

References

7. 8. 9. 10. 11. 12.

13.

14.

15. 16. 17. 18. 19.

20.

ip t

cr

us

6.

an

5.

M

4.

d

3.

te

2.

Albert, A.P., et al., Multiple activation mechanisms of store-operated TRPC channels in smooth muscle cells. J Physiol, 2007. 583(Pt 1): p. 25-36. Emptage, N.J., C.A. Reid, and A. Fine, Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release. Neuron, 2001. 29(1): p. 197-208. Ong, H.L. and I.S. Ambudkar, The dynamic complexity of the TRPC1 channelosome. Channels (Austin), 2011. 5(5): p. 424-31. Parekh, A.B. and J.W. Putney, Jr., Store-operated calcium channels. Physiol Rev, 2005. 85(2): p. 757-810. Venkatachalam, K. and C. Montell, TRP channels. Annu Rev Biochem, 2007. 76: p. 387417. Zhou, F.W., S.G. Matta, and F.M. Zhou, Constitutively active TRPC3 channels regulate basal ganglia output neurons. J Neurosci, 2008. 28(2): p. 473-82. Lemos, V.S., et al., Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells. Biochem Biophys Res Commun, 2007. 352(1): p. 130-4. Hoth, M. and R. Penner, Calcium release-activated calcium current in rat mast cells. J Physiol, 1993. 465: p. 359-86. Hoth, M., C. Fasolato, and R. Penner, Ion channels and calcium signaling in mast cells. Ann N Y Acad Sci, 1993. 707: p. 198-209. Parekh, A.B. and R. Penner, Store depletion and calcium influx. Physiol Rev, 1997. 77(4): p. 901-30. Zhang, X., et al., Complex role of STIM1 in the activation of store-independent Orai1/3 channels. J Gen Physiol, 2014. 143(3): p. 345-59. Thompson, J.L. and T.J. Shuttleworth, A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel. Channels (Austin), 2012. 6(5): p. 370-8. Mignen, O., J.L. Thompson, and T.J. Shuttleworth, Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels. J Physiol, 2008. 586(1): p. 185-95. Liu, X., K. Groschner, and I.S. Ambudkar, Distinct Ca(2+)-permeable cation currents are activated by internal Ca(2+)-store depletion in RBL-2H3 cells and human salivary gland cells, HSG and HSY. J Membr Biol, 2004. 200(2): p. 93-104. Wes, P.D., et al., TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci U S A, 1995. 92(21): p. 9652-6. Zhu, X., et al., Molecular cloning of a widely expressed human homologue for the Drosophila trp gene. FEBS Lett, 1995. 373(3): p. 193-8. Ambudkar, I.S., et al., TRPC1: the link between functionally distinct store-operated calcium channels. Cell Calcium, 2007. 42(2): p. 213-23. Beech, D.J., TRPC1: store-operated channel and more. Pflugers Arch, 2005. 451(1): p. 5360. Brazer, S.C., et al., Caveolin-1 contributes to assembly of store-operated Ca2+ influx channels by regulating plasma membrane localization of TRPC1. J Biol Chem, 2003. 278(29): p. 27208-15. Cheng, K.T., et al., Local Ca(2)+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca(2)+ signals required for specific cell functions. PLoS Biol, 2011. 9(3): p. e1001025.

Ac ce p

1.

23 Page 25 of 31

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

40.

41. 42. 43. 44.

ip t

cr

us

26.

an

25.

M

24.

d

23.

te

22.

Smyth, J.T., et al., Dissociation of regulated trafficking of TRPC3 channels to the plasma membrane from their activation by phospholipase C. J Biol Chem, 2006. 281(17): p. 11712-20. Odell, A.F., J.L. Scott, and D.F. Van Helden, Epidermal growth factor induces tyrosine phosphorylation, membrane insertion, and activation of transient receptor potential channel 4. J Biol Chem, 2005. 280(45): p. 37974-87. Tian, D., et al., Antagonistic regulation of actin dynamics and cell motility by TRPC5 and TRPC6 channels. Sci Signal, 2010. 3(145): p. ra77. Cayouette, S., et al., Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation. J Biol Chem, 2004. 279(8): p. 7241-6. Maxfield, F.R. and T.E. McGraw, Endocytic recycling. Nat Rev Mol Cell Biol, 2004. 5(2): p. 121-32. Doherty, G.J. and H.T. McMahon, Mechanisms of endocytosis. Annu Rev Biochem, 2009. 78: p. 857-902. Taguchi, T., Emerging roles of recycling endosomes. J Biochem, 2013. 153(6): p. 505-10. Grant, B.D. and J.G. Donaldson, Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol, 2009. 10(9): p. 597-608. Hong, W. and S. Lev, Tethering the assembly of SNARE complexes. Trends Cell Biol, 2013. Stenmark, H., Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol, 2009. 10(8): p. 513-25. Gerasimenko, J.V., et al., Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr Biol, 1998. 8(24): p. 1335-8. Lloyd-Evans, E., et al., Endolysosomal calcium regulation and disease. Biochem Soc Trans, 2010. 38(6): p. 1458-64. Zerial, M. and H. McBride, Rab proteins as membrane organizers. Nat Rev Mol Cell Biol, 2001. 2(2): p. 107-17. Maxfield, F.R. and D.J. Yamashiro, Endosome acidification and the pathways of receptormediated endocytosis. Adv Exp Med Biol, 1987. 225: p. 189-98. Jean, S. and A.A. Kiger, Coordination between RAB GTPase and phosphoinositide regulation and functions. Nat Rev Mol Cell Biol, 2012. 13(7): p. 463-70. Huotari, J. and A. Helenius, Endosome maturation. EMBO J, 2011. 30(17): p. 3481-500. Altschuler, Y., C. Hodson, and S.L. Milgram, The apical compartment: trafficking pathways, regulators and scaffolding proteins. Curr Opin Cell Biol, 2003. 15(4): p. 423-9. Yuan, J.P., et al., Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell, 2003. 114(6): p. 777-89. Kim, J.Y., et al., Homer 1 mediates store- and inositol 1,4,5-trisphosphate receptordependent translocation and retrieval of TRPC3 to the plasma membrane. J Biol Chem, 2006. 281(43): p. 32540-9. Pani, B., et al., Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1. Proc Natl Acad Sci U S A, 2009. 106(47): p. 20087-92. Nabi, I.R., Cavin fever: regulating caveolae. Nat Cell Biol, 2009. 11(7): p. 789-91. Vinten, J., et al., Identification of a major protein on the cytosolic face of caveolae. Biochim Biophys Acta, 2005. 1717(1): p. 34-40. Pani, B. and B.B. Singh, Lipid rafts/caveolae as microdomains of calcium signaling. Cell Calcium, 2009. 45(6): p. 625-33. Lockwich, T.P., et al., Assembly of Trp1 in a signaling complex associated with caveolinscaffolding lipid raft domains. J Biol Chem, 2000. 275(16): p. 11934-42.

Ac ce p

21.

24 Page 26 of 31

51.

52. 53.

54. 55.

56.

57. 58. 59. 60.

61.

62.

ip t

cr

us

50.

an

49.

M

48.

d

47.

te

46.

Bergdahl, A., et al., Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1. Circ Res, 2003. 93(9): p. 83947. Kannan, K.B., D. Barlos, and C.J. Hauser, Free cholesterol alters lipid raft structure and function regulating neutrophil Ca2+ entry and respiratory burst: correlations with calcium channel raft trafficking. J Immunol, 2007. 178(8): p. 5253-61. Formigli, L., et al., Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation. J Cell Sci, 2009. 122(Pt 9): p. 1322-33. Brownlow, S.L. and S.O. Sage, Transient receptor potential protein subunit assembly and membrane distribution in human platelets. Thromb Haemost, 2005. 94(4): p. 839-45. Pani, B., et al., Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1. J Cell Sci, 2012. Kwiatek, A.M., et al., Caveolin-1 regulates store-operated Ca2+ influx by binding of its scaffolding domain to transient receptor potential channel-1 in endothelial cells. Mol Pharmacol, 2006. 70(4): p. 1174-83. Sundivakkam, P.C., et al., Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells. Am J Physiol Cell Physiol, 2009. 296(3): p. C403-13. Ambudkar, I.S., Cellular domains that contribute to Ca2+ entry events. Sci STKE, 2004. 2004(243): p. pe32. Ong, H.L. and I.S. Ambudkar, Role of lipid rafts in the regulation of store-operated Ca2+ channels, in Cholesterol regulation of ion channels and receptors, I. Levitan and F. Barrantes, Editors. 2012, John Wiley & Sons: Hoboken, N.J. p. 69-90. Zeng, W., et al., STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol Cell, 2008. 32(3): p. 439-48. Pani, B., et al., Lipid Rafts Determine Clustering of STIM1 in Endoplasmic ReticulumPlasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE). J Biol Chem, 2008. 283(25): p. 17333-40. Ambudkar, I.S., et al., Function and regulation of TRPC1: contribution to Ca2+ entry activated by store depletion, in Store-operated Ca2+ entry (SOCE) pathways, K. Groschner, W.F. Graier, and C. Romanin, Editors. 2011, Springer: New York. p. 73-92. Murata, T., et al., Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells. J Biol Chem, 2007. 282(22): p. 16631-43. Hong, J.H., et al., Polarized but differential localization and recruitment of STIM1, Orai1 and TRPC channels in secretory cells. Traffic, 2011. 12(2): p. 232-45. Ambudkar, I.S., Ca signaling and regulation of fluid secretion in salivary gland acinar cells. Cell Calcium, 2014. Stiber, J.A., et al., Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity. Mol Cell Biol, 2008. 28(8): p. 2637-47. Mehta, D., et al., RhoA interaction with inositol 1,4,5-triphosphate receptor and transient receptor potential channel-1 regulates Ca2+ entry. The Journal of Biological Chemistry, 2003. 278(35): p. 33492 - 33500. Bollimuntha, S., E. Cornatzer, and B.B. Singh, Plasma membrane localization and function of TRPC1 is dependent on its interaction with beta-tubulin in retinal epithelium cells. Vis Neurosci, 2005. 22(2): p. 163-70.

Ac ce p

45.

25 Page 27 of 31

69.

70.

71.

72.

73. 74. 75. 76.

77. 78. 79. 80.

ip t

cr

us

68.

an

67.

M

66.

d

65.

te

64.

Sutton, K.A., et al., Enkurin is a novel calmodulin and TRPC channel binding protein in sperm. Dev Biol, 2004. 274(2): p. 426-35. Liman, E.R., D.P. Corey, and C. Dulac, TRP2: a candidate transduction channel for mammalian pheromone sensory signaling. Proc Natl Acad Sci U S A, 1999. 96(10): p. 5791-6. Lucas, P., et al., A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: mechanism of pheromone transduction. Neuron, 2003. 40(3): p. 551-61. Jungnickel, M.K., et al., Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3. Nat Cell Biol, 2001. 3(5): p. 499-502. Mast, T.G., J.H. Brann, and D.A. Fadool, The TRPC2 channel forms protein-protein interactions with Homer and RTP in the rat vomeronasal organ. BMC Neurosci, 2010. 11: p. 61. Bandyopadhyay, B.C., et al., TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1. The Journal of Biological Chemistry, 2008. 283(47): p. 32821-30. Singh, B.B., et al., VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to agonist-stimulated Ca2+ influx. Mol Cell, 2004. 15(4): p. 635-46. Liu, X., et al., Molecular analysis of a store-operated and 2-acetyl-sn-glycerol-sensitive non-selective cation channel. Heteromeric assembly of TRPC1-TRPC3. J Biol Chem, 2005. 280(22): p. 21600-6. Lee, K.P., et al., Molecular Determinants Mediating Gating of Transient Receptor Potential Canonical (TRPC) Channels by Stromal Interaction Molecule 1 (STIM1). J Biol Chem, 2014. 289(10): p. 6372-82. Adebiyi, A., D. Narayanan, and J.H. Jaggar, Caveolin-1 assembles type 1 inositol 1,4,5trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells. J Biol Chem, 2011. 286(6): p. 4341-8. van Rossum, D.B., et al., Phospholipase Cgamma1 controls surface expression of TRPC3 through an intermolecular PH domain. Nature, 2005. 434(7029): p. 99-104. Patterson, R.L., et al., Phospholipase C-gamma: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci, 2005. 30(12): p. 688-97. Lussier, M.P., et al., RNF24, a new TRPC interacting protein, causes the intracellular retention of TRPC. Cell Calcium, 2008. 43(5): p. 432-43. Lockwich, T., et al., Stabilization of cortical actin induces internalization of transient receptor potential 3 (Trp3)-associated caveolar Ca2+ signaling complex and loss of Ca2+ influx without disruption of Trp3-inositol trisphosphate receptor association. J Biol Chem, 2001. 276(45): p. 42401-8. Kiselyov, K., et al., Protein-protein interaction and functionTRPC channels. Pflugers Arch, 2005. 451(1): p. 116-24. Lockwich, T., et al., Analysis of TRPC3-interacting proteins by tandem mass spectrometry. Journal of Proteome research, 2008. 7(3): p. 979-989. Lussier, M.P., et al., MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC. J Biol Chem, 2005. 280(19): p. 19393-400. Mery, L., et al., The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells. J Cell Sci, 2002. 115(Pt 17): p. 3497-508.

Ac ce p

63.

26 Page 28 of 31

87.

88. 89.

ip t

cr

us

86.

an

85.

M

84.

d

83.

te

82.

Tang, Y., et al., Association of mammalian Trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF. The Journal of Biological Chemistry, 2000. 275(48): p. 27559 - 27564. Goel, M., et al., Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase. Pflugers Arch, 2005. 451(1): p. 87-98. Greka, A., et al., TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nature Neuroscience, 2003. 6(8): p. 837 - 845. Bezzerides, V.J., et al., Rapid vesicular translocation and insertion of TRP channels. Nat Cell Biol, 2004. 6(8): p. 709-20. Bandyopadhyay, B.C., et al., Apical localization of a functional TRPC3/TRPC6-Ca2+signaling complex in polarized epithelial cells. Role in apical Ca2+ influx. J Biol Chem, 2005. 280(13): p. 12908-16. Cayouette, S., et al., Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6. Biochim Biophys Acta, 2010. 1803(7): p. 805-12. Monet, M., N. Francoeur, and G. Boulay, Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells. J Biol Chem, 2012. 287(21): p. 17672-81. Redondo, P.C., et al., A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets. J Physiol, 2004. 558(Pt 1): p. 99-109. Song, X., et al., Canonical transient receptor potential channel 4 (TRPC4) co-localizes with the scaffolding protein ZO-1 in human fetal astrocytes in culture. Glia, 2005. 49(3): p. 418-29.

Ac ce p

81.

27 Page 29 of 31

us

cr

i

Figures

Ac

ce

pt

ed

M

an

Figure 1

Page 30 of 31

i cr us

Ac

ce

pt

ed

M

an

Figure 2

Page 31 of 31