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.

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.

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice.

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells.

Invertebrate TRP proteins as functional models for mammalian channels.

Transient receptor potential (TRP) channels constitute a large and diverse family of channel proteins that are expressed in many tissues and cell types in both vertebrates and invertebrates. While the biophysical features of many of the mammalian TRP channels have been described, relatively little is known about their biological roles. Invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo.

Decavanadate modulates gating of TRPM4 cation channels.

We have tested the effects of decavanadate (DV), a compound known to interfere with ATP binding in ATP-dependent transport proteins, on TRPM4, a Ca(2+)-activated, voltage-dependent monovalent cation channel, whose activity is potently blocked by intracellular ATP(4-). Application of micromolar Ca(2+) concentrations to the cytoplasmic side of inside-out patches led to immediate current activation followed by rapid current decay, which can be explained by an at least 30-fold decreased apparent affinity for Ca(2+). Subsequent application of DV (10 microm) strongly affected the voltage-dependent gating of the channel, resulting in large sustained currents over the voltage range between -180 and +140 mV.

Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G.

Arachidonic acid (AA) modulates T-type Ca(2+) channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type Ca(2+) channel alpha(1G) heterologously expressed in HEK-293 cells.

The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels.

The mammalian sensory system is capable of discriminating thermal stimuli ranging from noxious cold to noxious heat. Principal temperature sensors belong to the TRP cation channel family, but the mechanisms underlying the marked temperature sensitivity of opening and closing ('gating') of these channels are unknown. Here we show that temperature sensing is tightly linked to voltage-dependent gating in the cold-sensitive channel TRPM8 and the heat-sensitive channel TRPV1.

[ATP release pathways in vascular endothelial cells].

Vascular endothelial cells regulate vascular tonus, growth, and angiogenesis in response to mechanical stresses. ATP release is one of well-known mechanosensitive responses in endothelial cells. Released ATP induces Ca(2+) responses and nitric oxide production in neighboring cells in an auto/paracrine manner.

Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b.

TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca(2+)-activated but Ca(2+)-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca(2+) concentrations applied at the inner side, [Ca(2+)](i), activated TRPM4 with an EC(50) value of 0.

Outer pore architecture of a Ca2+-selective TRP channel.

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis.

TRPV4 calcium entry channel: a paradigm for gating diversity.

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as Ca2+ entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4alpha-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids.

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.

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 4alphaPDD. An endogenous ligand for this channel has not yet been identified.

Voltage dependence of the Ca2+-activated cation channel TRPM4.

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches.

Modulation of TRPV4 gating by intra- and extracellular Ca2+.

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4alphaPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+](e)) reduced the current amplitude and accelerated its decay.

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1).

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.

Pore structure influences gating properties of the T-type Ca2+ channel alpha1G.

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I-IV of the alpha1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the alpha1G subunit (CaV3.

Transient receptor potential channels in endothelium: solving the calcium entry puzzle?

Many endothelial cell (EC) functions depend on influx of extracellular Ca2+, which is triggered by a variety of mechanical and chemical signals. Here, we discuss possible pathways for this Ca2+ entry. The superfamily of cation channels derived from the "transient receptor potential" (TRP) channels is introduced.

The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation.

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector.

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6.

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+).

Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells.

We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25 degrees C activated currents and increased [Ca(2+)](i) in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4alphaPDD (alpha-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance.

ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells.

We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.

Molecular determinants of permeation through the cation channel TRPV4.

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore.

Fast and slow inactivation kinetics of the Ca2+ channels ECaC1 and ECaC2 (TRPV5 and TRPV6). Role of the intracellular loop located between transmembrane segments 2 and 3.

The Ca(2+) channels ECaC1 and ECaC2 (TRPV5 and TRPV6) share several functional properties including permeation profile and Ca(2+)-dependent inactivation. However, the kinetics of ECaC2 currents notably differ from ECaC1 currents. The initial inactivation is much faster in ECaC2 than in ECaC1, and the kinetic differences between Ca(2+) and Ba(2+) currents are more pronounced for ECaC2 than ECaC1.

RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells.

Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I(Cl,swell). However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect.