Guy Droogmans

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.

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption.

Mg2+ is an essential ion involved in a multitude of physiological and biochemical processes and a major constituent of bone tissue. Mg2+ homeostasis in mammals depends on the equilibrium between intestinal Mg2+ absorption and renal Mg2+ excretion, but little is known about the molecular nature of the proteins involved in the transepithelial transport of Mg2+ in these organs. Recently, it was shown that patients with mutations in TRPM6, a member of the transient receptor potential family of cation channels, suffer from hypomagnesemia with secondary hypocalcemia (HSH) as a result of impaired renal and/or intestinal Mg2+ handling. Here, we show that TRPM6 is specifically localized along the apical membrane of the renal distal convoluted tubule and the brush-border membrane of the small intestine, epithelia particularly associated with active Mg2+ (re)absorption. In kidney, parvalbumin and calbindin-D28K, two divalent-binding proteins, are co-expressed with TRPM6 and might function as intracellular Mg2+ buffers in the distal convoluted tubule. Heterologous expression of wild-type TRPM6 but not TRPM6 mutants identified in HSH patients induces a Mg2+- and Ca2+-permeable cation channel tightly regulated by intracellular Mg2+ levels. The TRPM6-induced channel displays strong outward rectification, has a 5-fold higher affinity for Mg2+ than for Ca2+, and is blocked in a voltage-dependent manner by ruthenium red. Our data indicate that TRPM6 comprises all or part of the apical Mg2+ channel of Mg2+-absorbing epithelia.

Heat activation of TRPM5 underlies thermal sensitivity of sweet taste

TRPM5, a cation channel of the TRP superfamily, is highly expressed in taste buds of the tongue, where it has a key role in the perception of sweet, umami and bitter tastes. Activation of TRPM5 occurs downstream of the activation of G-protein-coupled taste receptors and is proposed to generate a depolarizing potential in the taste receptor cells. Factors that modulate TRPM5 activity are therefore expected to influence taste. Here we show that TRPM5 is a highly temperature-sensitive, heat-activated channel: inward TRPM5 currents increase steeply at temperatures between 15 and 35 °C. TRPM4, a close homologue of TRPM5, shows similar temperature sensitivity. Heat activation is due to a temperature-dependent shift of the activation curve, in analogy to other thermosensitive TRP channels. Moreover, we show that increasing temperature between 15 and 35 °C markedly enhances the gustatory nerve response to sweet compounds in wild-type but not in Trpm5 knockout mice. The strong temperature sensitivity of TRPM5 may underlie known effects of temperature on perceived taste in humans, including enhanced sweetness perception at high temperatures and ‘thermal taste’, the phenomenon whereby heating or cooling of the tongue evoke sensations of taste in the absence of tastants.

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.

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.

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.

Regulation of a swelling‐activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins

1 The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling‐activated Cl− current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole‐cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5 % (mild hypotonicity) or 25 % (strong hypotonicity). 2 The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 ± 0.2, 61.4 ± 1.7 and 62.9 ± 1.3μM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3 The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 μM) and dephostatin (20 μM) potentiated ICl,swell activated by mild hypotonicity by 47 ± 9 and 69 ± 15 %, respectively. 4 Intracellular perfusion with GTPγS (100 μM) transiently activated a Cl− current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity‐induced cell shrinkage completely inhibited the GTPγS‐activated Cl− current. 5 Intracellular perfusion with GDPβS (1 mM) caused a time‐dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6 Our results demonstrate that the activity of endothelial swelling‐activated Cl− channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells

1 We combined patch clamp and fura‐2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2 ATP, bradykinin and intracellular InsP3 activated a non‐selective cation current (IhTRP3) in htrp3‐transfected CPAE cells but not in non‐transfected wild‐type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3‐transfected cells than in control CPAE cells. 3 The permeability for monovalent cations was PNa > PCs≈PK >> PNMDG and the ratio PCa/PNa was 1·62 ± 0·27 (n= 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist‐activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24·4 ± 0·7 μM). 4 The single‐channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5 Thapsigargin and 2,5‐di‐tert‐butyl‐1,4‐benzohydroquinone (BHQ), inhibitors of the organellar Ca2+‐ATPase, failed to activate IhTRP3. U‐73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor‐sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6 It is concluded that hTRP3 forms non‐selective plasmalemmal cation channels that function as a pathway for agonist‐induced Ca2+ influx.

Differential expression of volume-regulated anion channels during cell cycle progression of human cervical cancer cells

1 This study investigated the volume‐regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. 2 Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I− > Br− > Cl−. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 μm. 3 Fluorescence‐activated cell sorting (FACS) experiments showed that 59 ± 0.5, 5 ± 0.5 and 36 ± 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 μm) arrested 88 ± 1.4% of cells at the G0/G1 stage. 4 Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity‐induced current decreased from 48 ± 5.2 pA pF−1 at +100 mV in unsynchronized cells to 15 ± 2.6 pA pF−1 at +100 mV in aphidicolin‐treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re‐entry into the cell cycle and a concomitant recovery of VRAC density. 5 Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. 6 This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.