Regulation of the cellular localization and function of human transient receptor potential channel 1 by other members of the TRPC family
Introduction
Agonist-induced increments in intracellular calcium are a widespread phenomenon associated with a large number of cellular functions. The increments in cytosolic calcium may result from the entry of extracellular calcium, or from the release of intracellular calcium stores, like the endoplasmic reticulum (ER). Both, calcium influx and calcium release, are tightly coupled via the so-called store-operated calcium entry process (SOCE) [1]; the proteins responsible for such SOCE are believed to be members of the transient receptor potential (TRP) channel superfamily (for a recent review, see [2]).
All TRPs contain six putative transmembrane domains and are thought to assemble with other TRP proteins as homo- or heterotetramers to form cation channels. The functional properties of homo- versus heterotetramers have been poorly explored to this date. TRP is the second largest superfamily of channels, right after potassium channels. As we reported originally, Drosophila TRP proteins can function as SOCE channels when expressed in SF9 cells [3]; the finding of homologs of the TRP protein in mammals introduces the possibility that they could be responsible for SOCE in higher organisms, with mammal TRPC channels sharing the highest identity with Drosophila TRP channels. The results so far are conflicting; with some studies favoring the SOCE function of some TRPC proteins, while others finding that they can function as receptor-operated channels mostly insensitive to store depletion. It is generally accepted that all TRPCs have overlapping binding sites for Calmodulin (CaM) and the inositol(1,4,5)trisphosphate (IP3R) receptor, thus raising the possibility of modulation of their function by the filling state of the ER in certain conditions [4].
In particular, TRPC1 has been proposed to be responsible for SOCE in salivary gland cells [5], A549 [6], pulmonary artery cells [7], Chinese hamster ovary (CHO) cells [8] and vascular smooth muscle cells [9], although the mechanism of gating in these cells has not been elucidated [10]. However, there is also evidence that TRPC1 is insensitive to store depletion when expressed alone [11]. Native TRPC1 from CHO cells is activated both by store-depletion and agonists coupled to the IP3 cascade [8]. The activation requires association to IP3R and is modulated by calmodulin [8].
More recently, we have shown that TRPC1 can associate to the skeletal muscle, type 1 ryanodine receptor (RyR1) [12]. The foot structure from RyR1 is required for this functional association [12]. However, in these studies it was not determined if TRPC1 was associated to other TRPC proteins, forming heterotetramers.
According to others studies, TRPC1 may only form functional channels when expressed as heterotetramer, since the major localization of TRPC1 (as homotetramer) appears to be in intracellular compartments [13]. It is experimentally established that TRPC1 interacts with TRPC4/5 [14], and more recently, TRPC1 translocation into the plasma membrane has been shown to depend upon the expression of TRPC4 [15]. However, the details of such interactions have not been completely outlined.
Recently, new proteins (STIM1 and Orai) that seem to be necessary for SOCE, were identified using wide screening RNAi-based approaches. STIM1 seems to be a Ca2+-sensing protein located at the ER membrane (and possibly on the plasma membrane). STIM1 may modulate the activity of cationic channels at the plasma membrane in response to the filling state of the ER. It was recently found that STIM1 interacts with TRPC channels, thereby regulating their activity during SOCE [16]. Orai1 was identified as a component of the calcium release activated current (ICRAC), a particular form of SOCE [17]. It has been found also that Orai1 physically interacts with TRPC channels, conferring them sensitivity to store depletion [18]. It is thus possible than the activity of TRPC channels as SOCE not only depends on the interaction among members of the TRPC family, but on its association to other proteins as well.
To further study the events that allow TRPC1 translocation to the plasma membrane and their relevance to channel function, we generated N-terminal fusion proteins of TRPC1, TRPC4 and TRPC5 with fluorescent proteins CFP or YFP. These chimeric proteins allowed us to examine their cellular localization using confocal microscopy, and heterotetramer formation with co-immunoprecipitation studies.
In agreement with previous reports, we found that TRPC1 expression in CHO and HEK293 cells resulted in fluorescent fusion proteins localized mainly to the ER. However, when TRPC1 was co-expressed with either TRPC4 or TRPC5 abundant plasma membrane fluorescence was observed.
Whole-cell and single channel electrophysiological experiments further confirmed that the biophysical properties of TRPC5 were altered when co-expressed with TRPC1, suggesting the formation of TRPC1–TRPC5 heterotetramers at the plasma membrane. No whole-cell currents were detected when TRPC1 was expressed alone, as expected since most of the protein remained at the ER.
To explore the function of TRPC1 when expressed alone (when present in the ER) we produced a novel chameleon TRPC1 protein containing a FRET-based Ca2+-sensing domain, which allowed us to investigate TRPC1 activity in the ER.
With this chimeric protein, local changes in FRET induced by agonist stimulation could be detected. The chimeric protein did not detect [Ca2+] increments induced with ionophores, suggesting that the FRET-based Ca2+-sensing domain was detecting localized changes in [Ca2+], probably due to [Ca2+] traversing the TRPC1 pore. A similar chimeric construct containing a pore-mutated version of TRPC1, further confirmed that the FRET reported by the chimera was the result of calcium traversing the TRPC1 pore. The chimeric mutant was unable to respond with FRET to agonist stimulation.
All these results taken together strongly suggest that TRPC1 functions as a homotetramer calcium release channel (in the ER) or as an agonist-activated channel (heterotetramer with TRPC4 or TRPC5) at the plasma membrane. Thus, both TRPC4/TRPC5 control the localization and function of TRPC1 in the cell. These results have important implications to understand the complex association between Ca2+ influx and Ca2+ release from internal stores, while providing novel physiological significance to the TRPC homo–heterotetramer formation.
Section snippets
Reagents and solutions
All salts were analytical grade purchased from Sigma (St. Louis, MO). Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) were obtained also from Sigma. The FM646 fluorescent dye, brefeldin conjugates and CM-DiI were purchased from Invitrogen–Molecular Probes (Invitrogen, Carlsbad, CA). All TRPC antibodies used were purchased from Alomone Labs (Jerusalem, Israel).
Plasmids
Plasmids containing the GFP mutants CFP and EYFP were purchased from Clontech (Mountain View, CA). The plasmid containing
Exogenous TRPC1 is expressed almost exclusively on internal membranes in the cell
It has been reported that when expressed alone, TRPC1 stays in intracellular location, probably the ER [15]. To determine TRPC1 localization in two different cell lines, we transfected either CHO or HEK293 cells with the fusion protein CFP-TRPC1.
Fig. 1 shows representative cell fluorescence confocal studies illustrating CFP-TRPC1 localization in the cell. Expression of TRPC1 alone shows an almost exclusive intracellular localization (see also Supplemental Fig. 1). Using the selective ER marker,
Discussion
Over a decade has passed since the identification of the first two members from the TRP superfamily of cationic channels [3], [28]. In spite of hundreds of studies conducted to this date with the different members from this superfamily of channels, there are many unsolved questions about their regulation and mechanisms of activation. The TRPC family appears to be the most mysterious in this regard. TRPC1 is not an exception, there are many studies indicating that this channel functions as a
Conflict of interest
None.
Acknowledgments
This work was supported by a grant from Consejo Nacional de Ciencia y Tecnología CONACyT (D42469) and DGAPA IN204406 to LV. The authors thank Dr. A. Navarro for technical assistance. We greatly appreciate the technical assistance from the Microscopy and Molecular Biology units at the Instituto de Fisiología Celular, UNAM.
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