Substance P evokes cation currents through TRP channels in HEK293 cells

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.     

PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain

The subjective feeling of cold is mediated by the activation of TRPM8 channels in thermoreceptive neurons by cold or by cooling agents such as menthol. Here, we demonstrate a central role for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the activation of recombinant TRPM8 channels by both cold and menthol. Moreover, we show that Ca(2+) influx through these channels activates a Ca(2+)-sensitive phospholipase C and that the subsequent depletion of PI(4,5)P(2) limits channel activity, serving as a unique mechanism for desensitization of TRPM8 channels. Finally, we find that mutation of conserved positive residues in the highly conserved proximal C-terminal TRP domain of TRPM8 and two other family members, TRPM5 and TRPV5, reduces the sensitivity of the channels for PI(4,5)P(2) and increases inhibition by PI(4,5)P(2) depletion. These data suggest that the TRP domain of these channels may serve as a PI(4,5)P(2)-interacting site and that regulation by PI(4,5)P(2) is a common feature of members of the TRP channel family.     

Transient Receptor Potential (TRP) channels in T cells

The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases.     

The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry

Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.     

Regulation of transient receptor potential (TRP) channels by phosphoinositides

This review summarizes the modulation of transient receptor potential (TRP) channels, by phosphoinositides. TRP channels are characterized by polymodal activation and a surprising complexity of regulation mechanisms. Possibly, most if not all TRP channels are modulated by phosphoinositides. Modulation by phosphatidylinositol 4,5-biphosphate (PIP₂) has been shown in detail for TRP vanilloid (TRPV) 1, TRPV5, TRP melastatin (TRPM) 4, TRPM5, TRPM7, TRPM8, TRP polycystin 2, and the Drosophila TPR-like (TRPL) channels. This review describes mechanisms of modulation of TRP channels mainly by PIP₂ and discusses some future challenges of this fascinating topic.     

Gating of TRP channels: a voltage connection?

TRP channels represent the main pathways for cation influx in non-excitable cells. Although TRP channels were for a long time considered to be voltage independent, several TRP channels now appear to be weakly voltage dependent with an activation curve extending mainly into the non-physiological positive voltage range. In connection with this voltage dependence, there is now abundant evidence that physical stimuli, such as temperature (TRPV1, TRPM8, TRPV3), or the binding of various ligands (TRPV1, TRPV3, TRPM8, TRPM4), shift this voltage dependence towards physiologically relevant potentials, a mechanism that may represent the main functional hallmark of these TRP channels. This review discusses some features of voltage-dependent gating of TRPV1, TRPM4 and TRPM8. A thermodynamic principle is elaborated, which predicts that the small gating charge of TRP channels is a crucial factor for the large voltage shifts induced by various stimuli. Some structural considerations will be given indicating that, although the voltage sensor is not yet known, the C-terminus may substantially change the voltage dependence of these channels.     

Potential role of transient receptor potential (TRP) channels in bladder cancer cells

Transient receptor potential (TRP) channels play important roles in thermal, chemical, and mechanical sensation in various tissues. In this study, we investigated the differences in urothelial TRP channels between normal urothelial cells and bladder cancer cells. TRPV2 and TRPM7 expression levels and TRPV2 activator-induced intracellular Ca²⁺ increases were significantly higher, whereas TRPV4 expression and TRPV4 activator-induced intracellular Ca²⁺ increases were significantly lower in mouse bladder cancer (MBT-2) cells compared to normal mouse urothelial cells. The proliferation rate of MBT-2 cells overexpressing dominant-negative TRPV2 was significantly increased. In contrast, treatment with TRPV2 activators significantly decreased the proliferation rate. TRPM7-overexpressing MBT-2 cells proliferated more slowly, as compared to mock-transfected cells. Moreover, expression of dominant-negative TRPV2 significantly decreased plasma membrane Ca²⁺ permeability of MBT-2 cells as compared to that in mock-transfected cells. Increases in the expression of TRPV2 mRNA, immunoreactivity, and TRPV2 activator-induced intracellular Ca²⁺ were also observed in T24 human bladder cancer cells. These results suggested that TRPV2 and TRPM7 were functionally expressed in bladder cancer cells and served as negative regulators of bladder cancer cell proliferation, most likely to prevent excess mechanical stresses.     

The C-terminus of Kv7 channels: a multifunctional module

Kv7 channels (KCNQ) represent a family of voltage-gated K⁺ channels which plays a prominent role in brain and cardiac excitability. Their physiological importance is underscored by the existence of mutations in human Kv7 genes, leading to severe cardiovascular and neurological disorders such as the cardiac long QT syndrome and neonatal epilepsy. Kv7 channels exhibit some structural and functional features that are distinct from other Kv channels. Notably, the Kv7 C-terminus is long compared to other K⁺ channels and is endowed with characteristic structural domains, including coiled-coils, amphipatic α helices containing calmodulin-binding motifs and basic amino acid clusters. Here we provide a brief overview of current insights and as yet unsettled issues about the structural and functional attributes of the C-terminus of Kv7 channels. Recent data indicate that the proximal half of the Kv7 C-terminus associates with one calmodulin constitutively bound to each subunit. Epilepsy and long QT mutations located in this proximal region impair calmodulin binding and can affect channel gating, folding and trafficking. The distal half of the Kv7 C-terminus directs tetramerization, employing tandem coiled-coils. Together, the data indicate that the Kv7 C-terminal domain is a multimodular structure playing a crucial role in channel gating, assembly and trafficking as well as in scaffolding the channel complex with signalling proteins.     

Voltage is a partial activator of rat thermosensitive TRP channels

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μ m ), resiniferatoxin (5 μ m ) and menthol (6 m m ) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.     

Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid

We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X₁, P2X₂ and P2X₆ receptors were detected exclusively in vascular smooth muscle; P2X₅ and P2X₇ receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X₃, P2X₄ and P2X₇ receptors. Thyroid follicular cells displayed immunoreactivity for P2X₃, P2X₄ and P2X₅ receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.     

The history of TRP channels, a commentary and reflection

The transient receptor potential (TRP) family of cation channels has redefined our understanding of sensory physiology. In one animal or another, all senses depend on TRP channels. These include vision, taste, smell, hearing, and various forms of touch, including the ability to sense changes in temperature. The first trp gene was identified because it was disrupted in a Drosophila mutant with defective vision. However, there was no clue as to its biochemical function until the cloning, and analysis of the deduced amino acid sequence suggested that trp encoded a cation channel. This concept was further supported by subsequent electrophysiological studies, including alteration of its ion selectivity by an amino acid substitution within the pore loop. The study of TRP channels emerged as a field with the identification of mammalian homologs, some of which are direct sensors of environmental temperature. At least one TRP channel is activated downstream of a thermosensory signaling cascade, demonstrating that there exist two modes of activation, direct and indirect, through which TRP channels respond to changes in temperature. Mutations in many TRP channels result in disease, including a variety of sensory impairments.     

Reconstitution in lipid bilayer of smooth muscle cation channels activated through a GTP-binding protein.

Reconstitution of G-protein-coupled receptor activated cation channels into the lipid bilayer was attempted with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca2+-dependent and ATP-sensitive K+ channels (Toro et al., 1990). Under Na+-rich conditions, incorporation of plasma membrane vesicles into the bilayer produced GTPgammaS (100 microM)-activatable channel activities that are inhibited by GDPbetaS (1 mM), sensitive to Ca2+ and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied in a manner dependent on Na+ concentration, but not affected by Cl-. These results strongly indicate that the reconstituted channels activated by GTPgammaS belong to a class of voltage-dependent, Ca2+-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also suggest potential usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle.     

Single-channel currents through transient-receptor-potential-like (TRPL) channels

Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between -120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and by a rise of intracellular Ca2+ (>2 microM). Constitutively active TRPL channels depolarized the host cells to -10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.     

Immunohistochemical identification of cells expressing ATP‐gated cation channels (P2X receptors) in the adult rat thyroid

We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X‐immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X₁, P2X₂ and P2X₆ receptors were detected exclusively in vascular smooth muscle; P2X₅ and P2X₇ receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X₃, P2X₄ and P2X₇ receptors. Thyroid follicular cells displayed immunoreactivity for P2X₃, P2X₄ and P2X₅ receptors. No immunostaining for P2X receptors was observed on C‐cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.     

Tricho-rhino-phalangeal syndrome type I (TRP I) due to an apparently balanced translocation involving 8q24

Tricho-rhino-phalangeal (TRP) syndromes type I and II are caused by a defective gene located on chromosome 8q24.1. We report a family with 2 sibs affected with TRP type I in combination with an apparently balanced chromosome (8;18) translocation involving 8q24.11. It is very likely that the 8q24 translocation breakpoint is physically linked to the TRP gene(s), thereby facilitating future efforts to clone the TRP gene(s). © 1993 Wiley-Liss, Inc.     

Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels

Permeabilities to glycerol and small non-electrolytes of three Aquaporin 1 CHIP (AQP1) water channels were measured in AQP1 cRNA-injected Xenopus laevis oocytes and in human AQP1 channels reconstituted in proteoliposomes. By an osmotic swelling assay, significant increases of ethylene glycol, glycerol and 1,3-propanediol apparent permeability coefficients (P_solutes) were found in oocytes expressing human, rat and frog AQP1. p-Chloromercuribenzene sulphonate (PCMBS) and CuSO₄ inhibited, by 95% and 58% respectively, apparent glycerol permeability (P _gly) in oocytes expressing human AQP1. pCMBS inhibition was reversed by -mercaptoethanol and CuSO₄ inhibition was partly reversed by the Cu²⁺-binding peptide Gly-Gly-His. Tritiated glycerol uptakes confirmed the augmented P _gly value of AQP1 cRNA-injected oocytes. In contrast, no increases of urea, meso-erythritol, D- or L-threitol, xylitol and mannitol uptakes were detected. Stopped-flow light scattering experiments performed with human AQP1 proteoliposomes also revealed a much greater increase of P _gly than did those with protein-free liposomes; the initial rate of proteoliposomes also swelling was inhibited by 96.2% with HgCl₂ and by 72.5% with CuSO₄. In AQP1 cRNA-injected oocytes and in proteoliposomes, the value of the glycerol reflection coefficient was 0.74–0.80, indicating that water and glycerol share the same pathway. All these results provide strong evidence that water and certain small solutes permeate the AQP1 channels expressed at the surface of X. laevis oocytes or reconstituted in proteoliposomes. The urea exclusion suggests that the selectivity of the AQP1 channels not only depends on the size of the solutes but probably also on their flexibility and their ability to form H-bonds.     

Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volume-regulated anion currents in HEK-293 cells

The detection of changes in volume and osmolality is an essential function in vertebrate cells. A novel member of the transient receptor potential (trp) family of ion channels, which is sensitive to changes in cell volume, has been described recently. Heterologous expression of TRP12 in HEK cells resulted in the appearance of a swelling-activated cation current. The permeability sequence of this cation current for various monovalent cations, as determined from shifts in reversal potential upon extracellular cation substitution, was PK>PCs>PNa>PLi, corresponding to an Eisenman-IV sequence characteristic for a weak-field-strength site. Surprisingly, over-expression of this channel in HEK cells was accompanied by a dramatic down-regulation of the volume-regulated anion channel (VRAC), which is activated by cell swelling in non-transfected cells. In contrast to VRAC, TRP12 could not be activated at constant volume by a reduction of intracellular ionic strength or by intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate (GTPgammaS). The kinetic and pharmacological profile of VRAC and TRP12 currents were also different.     

The tricho-rhino-phalangeal syndrome(s): Chromosome 8 long arm deletion: Is there a shortest region of overlap between reported cases? TRP I and TRP II syndromes: Are they separate entities?

Critical cytogenetic (re)evaluation of 2 of our own cases of tricho-rhino-phalangeal syndrome II (TRP II), or Langer-Giedion syndrome (LGS), and 10 cases from the literature, suggests that the shortest region of overlap of the 8q deletion is a part of band q24. 1. This region is assumed to be causally related to this syndrome, and possibly also to TRP I syndrome which, therefore, may not be a causally separate entity.     

Poorly selective cation channels in the skin of the larval frog (Stage≤XIX)

The abdominal skin of bullfrog larvae (Rana catesbeiana) was placed in an Ussing-type chamber, and its transepithelial electrical parameters were recorded with mucosal solutions of different ionic composition. With K⁺-like cations (K⁺, NH ₄ ⁺ , Rb⁺, Cs^–) the power spectra of the fluctuations in short-circuit current displayed a Lorentzian component (f _c =30–40 Hz). The relaxation noise could be suppressed by addition of the K⁺-channel blockers Ba²⁺ and TEA to the mucosal solution. Also, in presence of the ionophore antibiotic nystatin the Lorentzian noise was abolished. The Na⁺-channel probes amiloride and benzimidazolyl-2-guanidine (BIG) both enhanced the relaxation noise obtained with the K⁺-like cations but, with Na⁺, and Li⁺, also caused the rise of a relaxation component above the background noise. In presence of amiloride or BIG, the addition of Ba²⁺, TEA and nystatin still abolished the Lorentzian noise. It can be concluded that the relaxation-noise source is located in the apical cell membranes of the tadpole skin. These spontaneously fluctuating cation channels do not seem to strictly discriminate between K⁺-like ions (K⁺, NH ₄ ⁺ , Rb⁺, Cs⁺) and Na⁺-like ions (Na⁺, Li⁺). On the other hand, well-known specific probes for K⁺ channels (Ba²⁺, TEA) and for Na⁺ channels (amiloride, BIG) interact with this apical cation channel. It is possible that the poorly selective channel plays a role in the ontogenesis of the specific Na⁺ transport in the maturing frog skin.