Jean Prenen

Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels

TRPV4 is a widely expressed cation channel of the ‘transient receptor potential’ (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4αPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5′,6′-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.

Bimodal Action of Menthol on the Transient Receptor Potential Channel TRPA1

TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities.

Homo‐ and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6

The molecular assembly of the epithelial Ca2+ channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85–100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co‐expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd2+ sensitivity and voltage‐dependent gating. Immuno precipitations using membrane fractions from oocytes co‐expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca2+ channels that varied with respect to Ca2+‐dependent inactivation, Ba2+ selectivity and pharmacological block. Thus, Ca2+‐transporting epithelia co‐expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca2+ transport kinetics.

Permeation and Gating Properties of the Novel Epithelial Ca 2! Channel*

The recently cloned epithelial Ca2+ channel (ECaC) constitutes the Ca2+influx pathway in 1,25-dihydroxyvitamin D3-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca2+ concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca2+ gradient, demonstrating the distinctive Ca2+ permeability and constitutive activation of ECaC. Cells dialyzed with 10 mm1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below −40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba2+or Sr2+ replaced Ca2+ and was absent in Ca2+-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratioP Ca:P Na calculated from the reversal potential at 30 mm[Ca2+] o was larger than 100. The divalent cation selectivity profile is Ca2+ > Mn2+ > Ba2+ ∼ Sr2+. Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca2+ was replaced by Ba2+ and was virtually abolished if [Ca2+] o was lowered to 1 nm. In conclusion, ECaC is a Ca2+ selective channel, exhibiting Ca2+-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.

Functional expression of the epithelial Ca2+ channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex

TRPV5 and TRPV6 constitute the Ca2+ influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca2+ channels using yeast two‐hybrid and GST pull‐down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C‐terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull‐down and co‐immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5–S100A10 complex. Furthermore, the S100A10–annexin 2 pair colocalizes with the Ca2+ channels in TRPV5‐expressing renal tubules and TRPV6‐expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2‐specific small interfering RNA inhibited TRPV5 and TRPV6‐mediated currents in transfected HEK293 cells. In conclusion, the S100A10–annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

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.

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate.

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+‐activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+‐permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5‐biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside‐out patches and rundown of TRPM4 currents in whole‐cell patch‐clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channels Ca2+ sensitivity 100‐fold. Conversely, activation of the phospholipase C (PLC)‐coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C‐terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C‐terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C‐terminal PH domain in PIP2 action. PLC‐mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

CaT1 and the calcium release-activated calcium channel manifest distinct pore properties.

The calcium release-activated calcium channel (CRAC) is a highly Ca2+-selective ion channel that is activated on depletion of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705–709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na+ and Cs+; effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg2+ inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg2+-gated channel not directly related to CRAC.

Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4

TRPM4, a Ca2+-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca2+. The mechanisms underlying the alterations in Ca2+ sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca2+ sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca2+ sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca2+ dramatically reduced TRPM4 activation. We identified five Ca2+-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca2+ sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca2+ sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.

Function and expression of the epithelial Ca2+ channel family: comparison of mammalian ECaC1 and 2

1 The epithelial Ca2+ channel (ECaC) family represents a unique group of Ca2+‐selective channels that share limited homology to the ligand‐gated capsaicin receptors, the osmolarity‐sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated that this family is restricted to two members, ECaC1 and ECaC2 (also named CaT1). 2 RT‐PCR analysis demonstrated that the two channels are co‐expressed in calbindin‐D‐containing epithelia, including small intestine, pancreas and placenta, whereas kidney and brain only express ECaC1 and stomach solely ECaC2. 3 From an electrophysiological point of view, ECaC1 and ECaC2 are highly similar channels. Differences concern divalent cation permeability, the kinetics of Ca2+‐dependent inactivation and recovery from inactivation. 4 Ruthenium red is a potent blocker of ECaC activity. Interestingly, ECaC2 has a 100‐fold lower affinity for ruthenium red (IC50 9 ± 1 μm) than ECaC1 (IC50 121 ± 13 nm). 5 ECaCs are modulated by intracellular Mg2+ and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg2+ concentrations. 6 In conclusion, ECaC1 and ECaC2 are homologous channels, with an almost identical pore region. They can be discriminated by their sensitivity for ruthenium red and show differences in Ca2+‐dependent regulation.