Establishing life is a calcium-dependent TRiP: Transient receptor potential channels in reproduction

Calcium plays a key role in many different steps of the reproduction process, from germ cell maturation to placental development. However, the exact function and regulation of calcium throughout subsequent reproductive events remains rather enigmatic. Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. On the one hand, endometrial cell will undergo massive changes to support an implanting embryo, including stromal cell decidualization. On the other hand, trophoblast cells from the trophectoderm surrounding the inner cell mass will differentiate and acquire new functions such as hormone secretion, invasion and migration. The need for calcium in the different gestational processes implicates the presence of specialized ion channels to regulate calcium homeostasis. The superfamily of transient receptor potential (TRP) channels is a class of calcium permeable ion channels that is involved in the transformation of extracellular stimuli into the influx of calcium, inducing and coordinating underlying signaling pathways. Although the necessity of calcium throughout reproduction cannot be negated, the expression and functionality of TRP channels throughout gestation remains elusive. This review provides an overview of the current evidence regarding the expression and function of TRP channels in reproduction.     

The TRPM6 Kinase Domain Determines the Mg·ATP Sensitivity of TRPM7/M6 Heteromeric Ion Channels

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg²⁺) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg²⁺ and therefore unlikely to be active at physiological levels of [Mg²⁺]_i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.     

Cytoskeletal and scaffolding proteins as structural and functional determinants of TRP channels

Transient receptor potential (TRP) channels are six transmembrane-spanning proteins, with variable selectivity for cations, that play a relevant role in intracellular Ca^2 + homeostasis. There is a large body of evidence that shows association of TRP channels with the actin cytoskeleton or even the microtubules and demonstrating the functional importance of this interaction for TRP channel function. Conversely, cation currents through TRP channels have also been found to modulate cytoskeleton rearrangements. The interplay between TRP channels and the cytoskeleton has been demonstrated to be essential for full activation of a variety of cellular functions. Furthermore, TRP channels have been reported to take part of macromolecular complexes including different signal transduction proteins. Scaffolding proteins play a relevant role in the association of TRP proteins with other signaling molecules into specific microdomains. Especially relevant are the roles of the Homer family members for the regulation of TRPC channel gating in mammals and INAD in the modulation of Drosophila TRP channels. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Properties of the TRPML3 Channel Pore and Its Stable Expansion by the Varitint-Waddler-causing Mutation

TRPML3 is a H⁺-regulated Ca²⁺ channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca²⁺ to change its conductance and permeability, which appears to be mediated by trapping Ca²⁺ within the pore. The pore properties can be restored by strong depolarization or by conducting Na⁺ through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca²⁺. This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.     

The venerable inveterate invertebrate TRP channels

The transient receptor potential (TRP) superfamily is subdivided into four main classes of cation channels, TRPC, TRPV, TRPM and TRPN, each of which includes members in worms, flies, mice and humans. While the biophysical features of many of the mammalian channels have been described, relatively little is known concerning the biological roles of these channels. Forward genetic screens in Drosophila melanogaster and Caenorhabditis elegans have led to the identification of the founding members of each of these four subfamilies. Moreover, phenotypic analyses of invertebrate mutants have contributed greatly to our understanding of the roles of TRP proteins. A recurring theme is that many of these proteins function in sensory signaling processes ranging from vision to olfaction, osmosensation, light touch, social feeding, and temperature- and mechanically-induced nociception. In addition, at least one invertebrate TRP protein is required for cell division. As many of these functions may be conserved among the mammalian TRPs, the invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

瞬时受体电位通道研究进展

瞬时受体电位通道(TRP channels)是位于细胞膜上的一类重要的阳离子通道超家族.根据氨基酸序列的同源性,将已发现的28种哺乳动物,TRP通道分为:TRPC、TRPV、TRPM、TRPA、TRPP和TRPML 6个亚家族.所有的TRP通道都具有6次跨膜结构域.不同的TRP通道对钙离子和钠离子选择性不同.TRP通道分布广泛,调节机制各异,通过感受细胞内外环境的各种刺激,参与痛温觉、机械感觉、味觉的发生和维持细胞内外环境的离子稳态等众多生命活动.     

Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing a pathway for large and otherwise relatively impermeant molecules. Further, we have shown recently that these nonselective cation channels, when activated by capsaicin, are potently and reversibly blocked by external application of quaternary ammonium compounds and local anesthetics. Here we describe a novel phenomenon in transient receptor potential channel pharmacology whereby their expression levels in Xenopus laevis oocytes, as assessed by the magnitude of macroscopic currents, are negatively correlated with extracellular blocker affinity: small current densities give rise to nanomolar blockade by quaternary ammoniums and this affinity decreases linearly as current density increases. Possible mechanisms to explain these data are discussed in light of similar observations in other channels and receptors.     

Calcium channels and pumps in cancer: changes and consequences

Increases in intracellular free Ca(2+) play a major role in many cellular processes. The deregulation of Ca(2+) signaling is a feature of a variety of diseases, and modulators of Ca(2+) signaling are used to treat conditions as diverse as hypertension to pain. The Ca(2+) signal also plays a role in processes important in cancer, such as proliferation and migration. Many studies in cancer have identified alterations in the expression of proteins involved in the movement of Ca(2+) across the plasma membrane and subcellular organelles. In some cases, these Ca(2+) channels or pumps are potential therapeutic targets for specific cancer subtypes or correlate with prognosis.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

Furanocoumarins Are a Novel Class of Modulators for the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel

Furanocoumarin imperatorin is the major active component of Angelica dahurica root extracts, widely used in traditional medicine to treat headache, toothache, and orbital eye pain. In this study, we investigated the mechanisms that may underlie the pain-relieving effects of the compound. We found that imperatorin significantly inhibited formalin- and capsaicin-induced nocifensive responses but did not alter baseline thermal withdrawal thresholds in the rat. We established that imperatorin is a weak agonist of TRPV1, a channel implicated in detecting several noxious stimuli, exhibiting a 50% effective concentration (EC₅₀) of 12.6 ± 3.2 μm. A specific TRPV1 antagonist, JNJ-17203212 (0.5 μm), potently inhibited imperatorin-induced TRPV1 activation. Site-directed mutagenesis studies revealed that imperatorin most likely acted via a site adjacent to or overlapping with the TRPV1 capsaicin-binding site. TRPV1 recovery from desensitization was delayed in the presence of imperatorin. Conversely, imperatorin sensitized TRPV1 to acid activation but did not affect the current amplitude and/or the activation-inactivation properties of Na_v1.7, a channel important for transmission of nociceptive information. Thus, our data indicate that furanocoumarins represent a novel group of TRPV1 modulators that may become important lead compounds in the drug discovery process aimed at developing new treatments for pain management.     

Proteins modulating TRP channel function

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.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.