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Briefly review the recent structural work of transient receptor potential canonical (TRPC) ion channels by using electron cryo-microscopy (cryo-EM). The high resolution structures of TRPC3, TRPC4, TRPC5 and TRPC6 are discussed.

TRPC4 ion channel was reported to be regulated by small molecular inhibitors and calmodulin. We discuss these findings in the context of other members of TRPC subfamily modulated by different stimulants.

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic-pituitary-gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca2+ concentration via its own Ca2+ permeability and via the activation of voltage-gated Ca2+ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aβ42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aβ42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4β and the TRPC4β-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.

Calcium entry from the extracellular space into cells is an important signaling mechanism in both physiological and pathophysiological functions. In non-excitable cells, store-operated calcium (SOC) entry represents a principal mode of calcium entry. Activation of SOC entry in pulmonary artery endothelial cells leads to the formation of inter-endothelial cell gaps and subsequent endothelial barrier disruption. Regulation of endothelial SOC entry is poorly understood. In this work, we identify two large molecular weight immunophilins, FKBP51 and FKBP52, as novel regulators of SOC entry in endothelial cells. Using cell fractionation studies and immunocytochemistry we determined that a fraction of these largely cytosolic proteins localize to the plasma membrane where SOC entry channels are found. That FKBP51 and FKBP52 associate with SOC entry channel protein complexes was supported by co-precipitation of the immunophilins with TRPC4, a subunit of the calcium-selective, SOC entry channel ISOC. Dexamethasone-induced upregulation of FKBP51 expression in pulmonary artery endothelial cells reduced global SOC entry as well as ISOC. Similar results were observed when FKBP51 was over-expressed in an inducible HEK293 cell line. On the other hand, when FKBP52 was over-expressed SOC entry was enhanced. When expression of FKBP52 was inhibited, SOC entry was decreased. Collectively, our observations support regulatory roles for these large molecular weight immunophilins in which FKBP51 inhibits, whereas FKBP52 enhances, SOC entry in endothelial cells.

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca(2+)](i)). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different signaling pathways.

TRP channels are involved in different signaling cascades; TRP channels can be activated via hormones and neurotransmitter in a receptor/G-protein-mediated manner or by osmotic, thermic or mechanic stimuli. The overall functional role of TRP channels within these processes of hormonal cellular control, nociception or cellular calcium homeostasis is still unclear, as these complex processes often involve macromolecular structures. Whereas the integration of Drosophila TRP in the phototransduction process is becoming clear, the understanding of the participation of mammalian TRP channels in signal transduction complexes is only beginning. TRP channels have been demonstrated to interact with PDZ domain proteins, and both scaffold and regulatory function have been shown for INAD, the PDZ domain protein of the Drosophila phototransduction complex. In mammalian cells, the interaction of NHERF and TRPC4 has been shown and it is anticipated that NHERF may abolish the apparent store-dependent regulation of TRPC4 and TRPC5. Whereas TRP channels and PDZ domain proteins form permanent heterodimeric proteins, the interaction of calcium-binding proteins is dependent on the calcium concentration and is, therefore, dynamic. The prototype of calcium-binding protein used for experiments is calmodulin; whether or not calmodulin is also the natural interaction partner of TRP channels is an open question.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and thus contributes to many vasoproliferative retinopathies including retinopathy of prematurity. Based on the importance of canonical transient receptor potential (TRPC) channels in VEGF signaling, we firstly evaluated the expression of TRPC channels in mouse retina by reverse transcriptase-polymerase chain reaction. All seven TRPC channels were expressed in mouse retina. TRPC4 channels were chosen for further analysis based on their upregulation on hypoxic retina according to the GEO database under the identifier GSE19886. Interestingly, TRPC4 suppression by intravitreal injection of siRNA against mTRPC4 significantly inhibited retinal neovascularization. To further investigate the effect of TRPC4 suppression on neovascularization, human retina microvascular endothelial cells (HRMECs) that are responsible for initiating neovascularization in response to increased VEGF in OIR retina were transfected with siRNA against TRPC4. As we have expected, suppression of TRPC4 effectively inhibited VEGF-induced migration and tube formation as well. Further evaluation on VEGF signaling pathway by western blot analysis of signaling molecules discovered that VEGF-induced activation of ERK, p38 MAPK and AKT signaling pathways were inhibited by suppression of TRPC4. These findings suggest that suppression of TRPC4 could be an alternative therapeutic option for VEGF-induced retinal neovascularization.

Transient receptor potential canonical (TRPC) channels function as cation channels. In a previous study, we identified the molecular determinants involved in promoting TRPC subunit assembly. In the present study, we used size-exclusion chromatography assays to show that the N-terminus of TRPC4 can self-associate and form a tetramer in cellulo. We further showed that the N-terminus of TRPC4 self-associates via the ankyrin repeat domain and the region downstream from the coiled-coil domain. GST pull-down, yeast two-hybrid, and circular dichroism approaches demonstrated that both domains can self-associate. These findings indicated that the self-association of two distinct domains in the N-terminus of TRPC4 is involved in the assembly of the tetrameric channel.

Members of the Canonical Transient Receptor Potential (TRPC) family of ionic channels are able to form homo- and heterotetrameric channels. Depending on the study, TRPC1 has been detected on both the surface and inside the cell, probably in the endoplasmic reticulum (ER). Likewise, TRPC1 has been described both as a store-operated channel and as one unable to function when forming a homotetramer. It is possible that the apparent differences in the expression and function of TRPC1 are due to its association with other proteins, possibly from the same TRPC family. In the present study we used confocal microscopy and a fluorescently tagged TRPC1 to examine the localization of this protein when co-expressed with other members of the TRPC family. Whole-cell and single channel electrophysiological recordings were conducted to study the function of TRPC1 expressed alone or co-expressed with other members of the TRPC family. A FRET-based calcium sensor fused to TRPC1 was used to assess the functionality of the intracellular TRPC1. Our results showed that TRPC4 and TRPC5 were able to increase the amount of membrane-expressed TRPC1 as evaluated by confocal microscopy and patch clamp recordings. The FRET-based calcium sensor fused to TRPC1 strongly suggests that this protein forms ER-expressed functional homotetrameric channels activated by agonists coupled to the IP 3 cascade. These results indicate that TRPC1 is a multifunctional protein able to form intracellular calcium release channels when expressed alone, and plasma membrane channels when co-expressed with TRPC4 or TRPC5, but not TRPC3 or TRPC6. Both (ER and plasma membrane) forms of the channel are activated upon addition of agonists coupled to the IP 3 cascade.

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Forster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP3) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4−/− mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.

Ca2+ store depletion activates both Ca2+ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca2+ selective current, I(SOC). Indeed, I(SOC) is a relatively small inward Ca2+ current that exhibits an approximate +40mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I(SOC), although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I(SOC) requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca2+ store depletion and channel activation. Thus, at present findings indicate a TRPC4-protein 4.1 physical linkage regulates I(SOC) activation following Ca2+ store depletion.