Transient Receptor Potential Cation Channel, Subfamily C, Member 2, Pseudogene
The transient receptor potential (TRP) channels were first detected in a Drosophila photoreceptor mutant, where continuous light resulted in a transient voltage response. Further studies revealed that the channels had a profound selectivity for calcium. As the light response in the photoreceptors is evoked by activation of a G protein and phospholipase C, it was speculated that vertebrate homologs of the channels could exist and that these channels could participate in, e.g., receptor- and store-operated calcium entry in response to agonist stimulations. The first vertebrate homologs of TRP were subsequently detected in 1995, and further intense studies resulted in the detection of a total of 29 vertebrate (28 in humans) TRP isoforms. Of these isoforms, the canonical transient receptor potential channel (TRPC) subfamily is the isoform that has the highest degree of homology to the Drosophila TRP channels. The TRPC channels are nonselective cation channels with a slightly higher selectivity for calcium than for sodium and potassium and may assemble both as homomeric and heteromeric complexes. Furthermore, all TRPC channels are activated through various phospholipase C-activated downstream pathways. The family consists of seven members (TRPC1-7), is widely expressed, and participates in a multitude of calcium-dependent physiological and pathophysiological processes. Of all the seven different members of TRPC, TRPC2 (a pseudogene in human and catarrhine primates) is perhaps the least investigated. However, in many species (e.g., rodents), TRPC2 has a profound role in the olfactory system and potently regulates both male and female behavior (Birnbaumer 2009).
Physiology of TRPC2
Expression of full-length TRPC2 transcripts has been reported for numerous vertebrate species including mouse and rat, New World monkeys, marsupials, and zebrafish. In several other species, TRPC2 is a pseudogene, which contains stop codons within the open reading frame. This loss of TRPC2 gene expression has been linked with the loss of vomeronasal organ function (see Zufall 2014). Expression of the mouse TRPC2 gene has been studied the most, and several splice variants have been identified. Interestingly, one of these is a truncated, N-terminal protein (smTRPC2), which does not form a channel but instead regulates activity of the longer variants similarly to N-terminal splice variants of Drosophila TRP (for a detailed explanation on the molecular biology of the TRPC2 gene, see Yildirim and Birnbaumer 2007; Miller 2014).
Sequence analysis of TRPC2 predicts a six-transmembrane protein with pore region between transmembrane segment five and six. Both the N- and the C-terminus are in the cytosol and contain ankyrin repeats and calcium-calmodulin and inositol 1,4,5-trisphosphate receptor (IP3R) binding sites (Yildirim and Birnbaumer 2007). As TRPC2 is a pseudogene in, e.g., humans and catarrhine primates, TRPC2 is probably the least investigated TRPC channel. Thus, relatively little is known about its physiological significance and interaction with other calcium regulating signaling molecules. Functional channels have, however, been found in several species, including mice, rat, fish, frogs, salamanders, and the gray mouse lemur. TRPC2 has also been detected in several tissues, particularly in the vomeronasal organ (VNO) of rodents, but also in the testis, brain, heart, spleen, in erythroblast in mice, and in the rat thyroid. The highest expression is unequivocally in the rodent VNO (Miller 2014; Zufall 2014).
Two splice variants, TRPC2a (clone 14) and TRPC2b (clone 17), increased both store-operated and receptor-operated calcium entry when heterologously expressed in COS-M6 cells, whereas two novel variants of TRPC2, mTRPC2α and mTRPC2β, failed to enhance store-operated and receptor-operated calcium entry when expressed in HEK-293 cells. In the latter case, the channels were possibly retained in intracellular membranes and failed to reach the plasma membrane. Furthermore, in HEK cells, transiently expressing TRPC2 increased an ATP-evoked current. In rodent sperm, TRPC2 may be an important regulator of calcium homeostasis, in particular store-operated calcium entry, as an antibody against TRPC2 decreased thapsigargin and the egg zona pellucida 3 protein-evoked calcium entry in mouse sperm. This suggested that TRPC2 is important for fertility of mice. However, TRPC2 knockout animals are still fertile, rendering the importance of TRPC2 in sperm function unclear. Furthermore, in sensory neurons of the VNO in mice, the diacylglycerol (DAG)-induced inward currents were potently attenuated in TRPC2 knockout mice, compared with wild-type mice (Zufall 2014).
The electrophysiological characterization of the TRPC2 channel has been performed using vomeronasal neurons. Using inside-out patches from distal dendritic tips of these neurons revealed a -3.3 pA single-channel current (recorded at -80 mV) when stimulated with the DAG analog 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG). The slope conductance was 42 pS in a symmetrical 150 mM sodium chloride solution. Furthermore, the relative permeability (Pion) ratio between calcium and sodium (PCa/PNa) was 2.7 ± 0.7 in bi-ionic conditions (Lucas et al. 2003).
In murine hematopoietic HCD-57 cells, TRPC2 participates in the erythropoietin (EPO)-evoked calcium increase. This mechanism is mediated by a mechanism dependent on phospholipase Cγ and IP3 receptors. Together, these components form a signaling complex. Interestingly, TRPC2 depletion protects red blood cells from oxidative stress. Furthermore, co-expressing TRPC2 and TRPC6 attenuated the EPO-evoked TRPC2-dependent calcium entry in EPO-receptor transfected CHO-S cells. In addition, an N-terminal splice variant of TRPC2 (smTRPC2) attenuated EPO-evoked, TRPC2-dependent calcium entry (Miller 2014).
Recent investigations have shown that TRPC2 is also of importance in the rat thyroid, as it is expressed both in primary rat thyroid cells and in rat thyroid FRTL-5 cells. In the FRTL-5 cells, TRPC2 seems to be constitutively active, participating in the regulation of calcium homeostasis in the endoplasmic reticulum. Interestingly, the activity of TRPC2 seems to be regulated through a sphingosine-1-phosphate-mediated autocrine mechanism. The physiological importance of this mechanism is still unclear. Importantly, calcium entry through TRPC2 participates in the regulating of the TSH receptor expression and thyroglobulin maturation. In addition, TRPC2 participates in regulating iodine homeostasis by regulating the calcium-activated anion channel anoctamin-1 (ANO1/TMEM16A). TRPC2 is also important in regulating the migration, as well as proliferation, of FRTL-5 cells (see Törnquist et al. 2014).
Importance of TRPC2 in the Olfactory System
The most well-studied physiological function of TRPC2 is in the olfaction of rodents. Two different anatomical localizations for olfactory neurons have evolved, the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) in terrestrial animals. The sensory neurons in the VNO are responsible for detecting water-soluble pheromones and olfactory cues important for gender-specific behavior. Thus, ablation of the VNO resulted in marked changes in gender-specific behavior. As mentioned above, TRPC2 is highly expressed in sensory neurons in the VNO of several species, and the physiological importance of TRPC2 in the VNO has been extensively studied in rodents. Thus, a loss of TRPC2 expression in these neurons results in a severe reduction in the ability to detect pheromones, resulting in altered gender-specific behavior: in TRPC2 knockout mice, the response to pheromones in the urine was severely diminished or totally abolished, and the males were unable to recognize the difference between male and female mice (i.e., lack of aggressiveness against other males). Furthermore, the male mice showed sexual behavior (e.g., mounting) against other male mice. In contrast, female TRPC2 knockout mice showed male characteristic behaviors, such as mounting of wild-type female mice, pelvic thrust, and ultrasonic vocalizations. Female TRPC2 knockout mice also showed altered maternal behavior, e.g., they more easily abandoned their pups and lacked aggressiveness against intruding mice (Liman et al. 1999; Stowers et al. 2002; Dulac and Torello 2003; Zufall 2014).
In addition to mice, TRPC2 has also been detected in the olfactory systems of, e.g., zebrafish (Sato et al. 2005) and the frog Xenopus (Sansone et al. 2014). The physiological importance of TRPC2 in these species is, however, still not well understood but may be important in detecting amino acid odors. In the red-legged salamander (Plethodon shermani) (Kiemnec-Tyburczy et al. 2012), a fully terrestrial salamander that expresses TRPC2 in its VNO, chemical cues are important for both social and sexual behavior. It is plausible that TRPC2 in this species elicits a similar role as in rodents.
Although most investigations have shown the importance of TRPC2 in the VNO, recent investigations have indicated that TRPC2 is also expressed in the main olfactory epithelium (MOE). This has been shown not only in mice but also in, e.g., Xenopus and the mouse lemur (Sansone et al. 2014; Hohenbrink et al. 2014). The physiological significance of TRPC2 in the MOE in mice is still not understood, but may certainly increase the complexity of the role of TRPC2 in at least rodent olfaction. Furthermore, recent investigations have, however, challenged the notion that TRPC2 should be the main regulator of behavior in response to pheromones. This suggestion is drawn from the fact that TRPC2 knockout mice still show some function of the VNO. One possibility is an enhanced chloride conductance. It has been speculated that activation of a calcium-activated chloride channel (apparently anoctamin-1/TMEM16A) and an efflux of chloride ions depolarize neurons in the VNO. Furthermore, also the cyclic nucleotide-gated channel alpha 2 (CNGA2), the potassium channel SK3, and the inward rectifying potassium channel (GIRK) have been suggested to participate in pheromone sensing in rodents (Zufall 2014; Yu 2015).
Interacting Partners of TRPC2
TRPC2 interacts with several other proteins. In the C-terminus of the channel, calmodulin competes with the IP3 receptor for binding to the calmodulin- and IP3 receptor binding (CIRB) domain in a calcium-dependent manner, suggesting that calcium and calmodulin may have an inhibitory effect on TRPC2 through a negative feedback loop. Calmodulin (CaM) binds to both the N- and C-terminal regions (Tang et al. 2001).
As mentioned above, IP3R and PLCγ form a complex with TRPC2 to enable EPO-induced calcium entry in hematopoietic cells (Miller 2014). A complex between TRPC2 and the IP3R has also been shown in the VNO (Brann et al. 2002). Furthermore, stromal-interacting molecule 1 (STIM1), an important regulator of store-operated calcium entry in cells, binds to TRPC2 via its ezrin/radixin/moesin (ERM) domain (Huang et al. 2006). Furthermore, the N-terminal region of TRPC2 contains both enkurin and ankyrin binding domains (Liman et al. 1999; Sutton et al. 2004). The Homer 1 adaptor proteins, in particular Homer 1b/c and 3, co-immunoprecipitate with TRPC2 (Yuan et al. 2003), and the IP3R-associated protein junctate binds TRPC2 (Stamboulian et al. 2005). Furthermore, the chaperon receptor transporting protein 1 (RTP1) interacts with TRPC2 and enhances expression of TRPC2 in the plasma membrane. These examples suggest a complex array of interactions with TRPC2, thus increasing the versatility of channel regulation. Interestingly, TRPC2 seems not to interact with other TRPC channels to form heterotetrameric ion channels. However, in murine primary erythroblasts, an inhibitory interaction of TRPC6 with TRPC2 has been shown, and the N-terminal splice variant smTRPC2 attenuated calcium entry through full-length TRPC2 (Miller 2014).
Although TRPC2 is clearly less investigated compared with other TRPC channels, its importance in rodent olfaction is undisputable. Novel data suggest that TRPC2 may have a role in olfaction also in other species. Furthermore, the results showing that TRPC2 has a role in regulating calcium signaling, both in murine hematopoietic cells and in rat thyroid cells, indicates that the physiological importance of TRPC2 is not yet fully clarified. Thus, further research in other tissues than those described above may uncover exciting novel functions for this channel.
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