TRPM5 in the battle against diabetes and obesity.

TRPM5 is a non-selective monovalent cation channel activated by increases in intracellular Ca2+ . It has a distinct expression pattern: expression is detected in chemosensitive tissues from solitary chemosensory cells to the taste receptor cells and in pancreatic ß-cells. The role of TRPM5 has been investigated with the use of knockout mouse models. Trpm5-/- mice have a lack of type II taste perception and show reduced glucose-induced insulin secretion. Expression levels of TRPM5 are reduced in obese, leptin-signalling-deficient mice, and mutations in TRPM5 have been associated with type II diabetes and metabolic syndrome. In this review, we aim to give an overview of the activation, selectivity, modulation and physiological roles of TRPM5.

On the putative role of transient receptor potential cation channels in asthma.

The mammalian transient receptor potential (TRP) superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC ('Canonical'), TRPV ('Vanilloid'), TRPM ('Melastatin'), TRPP ('Polycystin'), TRPML ('Mucolipin') and the TRPA ('Ankyrin') groups. Increasing evidence has accumulated during the previous few years that links TRP channels to the cause of several diseases or to critically influence and/or determine their progress. This review focuses on the possible role of TRP channels in the aetiology of asthmatic lung disease.

Insights into TRPM4 function, regulation and physiological role.

In the current review we will summarise data from the recent literature describing molecular and functional properties of TRPM4. Together with TRPM5, these channels are up till now the only molecular candidates for a class of non-selective, Ca(2+)-impermeable cation channels which are activated by elevated Ca2+ levels in the cytosol. Apart from intracellular Ca2+, TRPM4 activation is also dependent on membrane potential. Additionally, channel activity is modulated by ATP, phosphatidylinositol bisphosphate (PiP2), protein kinase C (PKC) phosphorylation and heat. The molecular determinants for channel activation, permeation and modulation are increasingly being clarified, and will be discussed here in detail. The physiological role of Ca(2+)-activated non-selective cation channels is unclear, especially in the absence of gene-specific knock-out mice, but evidence indicates a role as a regulator of membrane potential, and thus the driving force for Ca2+ entry from the extracellular medium.

Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models.

In order to elucidate the functional role of TRPC genes, in vivo, the targeted inactivation of these genes in mice is an invaluable technique. In this review, we summarize the currently available results on the phenotype of TRPC-deficient mouse lines. The analysis of mice with targeted deletion in three TRPC genes demonstrates that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent Ca2+ entry channels in primary cells. Furthermore, from the deficits observed in these TRPC-deficient mouse lines a striking number of biological functions could already be ascribed to TRPC2, TRPC4, and TRPC6, not only on the cellular level but also for complex organ functions and integrative physiology. Accordingly, TRPC2 proteins are critically involved in pheromone sensing by neurones of the vomeronasal organ and, thereby, in the regulation of sexual and social behavior of mice, TRPC4 proteins are essential determinants of endothelial-dependent regulation of vascular tone, endothelial permeability, and neurotransmitter release from thalamic interneurones, and TRPC6 proteins are supposed to have a fundamental role in the regulation of smooth muscle tone in blood vessels and lung.

Current understanding of mammalian TRP homologues.

Calcium influx into the cell from the extracellular medium is crucial for important processes including muscle contraction, secretion and gene expression. This calcium influx is mainly mediated through calcium influx channels, which on the basis of their activation mechanism can be subdivided in voltage-gated calcium channels, which have already been thoroughly characterized and non-voltage-gated calcium permeable channels. This latter group includes ion channels activated by binding of extra and intracellular messengers, mechanical stress or depletion of intracellular calcium stores. Currently little molecular data is available concerning this class of calcium influx channels. However, recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels can function as calcium influx channels both in excitable and non-excitable tissues. On the basis of structural information the TRP family is subdivided in three main subfamilies: the TRPC (canonical) group, the TRPV (vanilloid) group and the TRPM (melastatin) group. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of tissues and species, including mammals, insects and yeast. This review summarizes the currently available information concerning members of the TRP family expressed in mammalian tissues.

Function and expression of the epithelial Ca(2+) channel family: comparison of mammalian ECaC1 and 2.

1. The epithelial Ca(2+) channel (ECaC) family represents a unique group of Ca(2+)-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 Ca(2+)-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 (IC(50) 9 +/- 1 microM) than ECaC1 (IC(50) 121 +/- 13 nM). 5. ECaCs are modulated by intracellular Mg(2+) and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg(2+) 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 Ca(2+)-dependent regulation.

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

The calcium release-activated calcium channel (CRAC) is a highly Ca(2+)-selective ion channel that is activated on depletion of inositol triphosphate (IP(3))-sensitive intracellular Ca(2+) 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 Mg(2+) inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg(2+)-gated channel not directly related to CRAC.

Pharmacological modulation of monovalent cation currents through the epithelial Ca2+ channel ECaC1.

1. The recent identification of the epithelial Ca(2+) channel, ECaC1, represents a major step forward in our knowledge of renal Ca(2+) handling. ECaC1 constitutes the rate-limiting apical Ca(2+) entry mechanism of active, transcellular Ca(2+) reabsorption. This unique highly selective Ca(2+) channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR). 2. We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca(2+) dependent inactivation of ECaC1. 3. Several modulators were tested, including inorganic cations, putative store-operated Ca(2+) entry (SOC) blockers, the vanilloid receptor (VR-1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red. 4. Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC(50) values of 111 nM and 1.3 microM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective. 5. The divalent cation current block profile for ECaC1 is Pb(2+)=Cu(2+) >Zn(2+) >Co(2+) >Fe(2+) with IC(50) values between 1 and approximately 10 microM. 6. In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca(2+) homeostasis could benefit from such developments.

Modulation of the epithelial Ca2+ channel ECaC by extracellular pH.

We investigated the effect of extracellular pH on whole-cell currents through the epithelial Ca2+ channel, ECaC, expressed in HEK 293 cells. Both mono- and divalent current densities were significantly smaller at pH 6.0 than at pH 7.4. At pH 8.5 they were slightly larger. Lowering extracellular pH enhanced the slow component of monovalent current activation at negative potentials but had no significant effect on the kinetics of Ca2+ currents. The kinetics of block of monovalent cation current by extracellular Mg2+ was significantly changed at high and low pH. The time constant of the time- and voltage-dependent current component during a voltage step to -140 mV was significantly larger at pH 8.5 than at pH 7.4. At pH 6.0 it was almost absent. The [Mg2+] inhibiting 50% of monovalent current through ECaC at pH 6.0 (IC50) was 323 +/- 23 microM (n = 8), compared with 62 +/- 9 microM (n = 4) at pH 7.4 and 38 +/- 4 microM (n = 8) at pH 8.5. The affinity of ECaC for Ca2+ was also affected by extracellular pH, shifting from 4.8 +/- 0.7 microM (n = 6) at pH 6.0 to 161 +/- 30 nM (n = 5) at pH 7.4 and 425 +/- 117 nM (n = 8) at pH 8.5.

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+.

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.

Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells.

We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.

Functional properties of the epithelial Ca2+ channel, ECaC.

ECaC is the first member of a new subfamily of Ca2+ channels embedded in the large TRPC family that includes numerous channel proteins. The channel has been proposed as the main gatekeeper of transcellular Ca2+ transport in kidney and intestine. The functional characterization of this channel is evolving rapidly and may have far reaching consequences for other channels of the TRPC family. The goal of this mini-review is to summarize the major functional and structural characteristics of ECaC, including (i) its proposed functional role, (ii) its channel structure and expression pattern, (iii) its main electrophysiological characteristics and (iv) its regulation.

The single pore residue Asp542 determines Ca2+ permeation and Mg2+ block of the epithelial Ca2+ channel.

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.

Whole-cell and single channel monovalent cation currents through the novel rabbit epithelial Ca2+ channel ECaC.

This study describes properties of monovalent cation currents through ECaC, a recently cloned epithelial Ca2+-permeable channel from rabbit. The kinetics of currents through ECaC was strongly modulated by divalent cations. Currents were inhibited in the presence of extracellular Ca2+. They showed an initial voltage-dependent decay in the presence of mM Mg2+ at hyperpolarizing steps in Ca2+-free solutions, which represents a voltage-dependent Mg2+ block through binding of Mg2+ to a site localized in the electrical field of the membrane (delta = 0.31) and a voltage-dependent binding constant (at 0 mV 3.1 mM Ca2+, obtained from a Woodhull type analysis). Currents were only stable in the absence of divalent cations and showed under these conditions a small time- and voltage-dependent component of activation. Single channel currents in cell-attached and inside-out patches had a conductance of 77.5 +/- 4.9 pS (n = 11) and reversed at +14.8 +/- 1. 6 11imV81i (n = 9) in the absence of divalent cations. The permeation sequence for monovalent cations through ECaC was Na+ > Li+ > K+ > Cs+ > NMDG+ which is identical to the Eisenmann sequence X for a strong field-strength binding site. It is concluded that the permeation profile of ECaC for monovalent cations suggests a strong field-strength binding site that may be involved in Ca2+ permeation and Mg2+ block.

Permeation and gating properties of the novel epithelial Ca(2+) channel.

The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-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 Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.

Characterisation of explanted endothelial cells from mouse aorta: electrophysiology and Ca2+ signalling.

We describe here the isolation and primary culture of endothelial cells from mouse aorta ("primary explant technique"). These cells provide an excellent model for functional studies in transgenic mice. The primary explant method delivers cells that grow out from small pieces of mouse aorta placed on Matrigel enriched with endothelial growth factors. Cells can be studied on the Matrigel after removing the pieces of aorta or after passages by using dispase and reseeding the cells on gelatine-coated cover-slips. Cells on Matrigel or from the first and second passages were characterised using the combined patch-clamp and fura-2 fluorescence methods. Cells had a mean membrane resting potential of -19+/-3 mV (n=21), a membrane capacitance of 49+/-5 pF (n=37) and a resting cytosolic free [Ca2+] ([Ca2+]i) of 103+/-8 nM (n=30). Adenosine 5'-triphosphate (ATP), acetylcholine and bradykinin, but not histamine, induced fast release of intracellular Ca2+ followed by a sustained rise in [Ca2+]i. Oscillations in [Ca2+]i were observed at lower agonist concentrations. In nearly all cells (93%, n=30), these agonists activated charybdotoxin-sensitive, Ca2+-activated K+ channels and induced hyperpolarisation. In 84% of the cells (n=32), an increase in [Ca2+]i also activated strongly outwards-rectifying Cl- channels. These activated slowly at positive potentials and inactivated rapidly at negative potentials. Increasing [Ca2+]i to 1 microM activated a non-selective cation channel in 86% of the cells (n=28). Each tested cell responded to a challenge with hypotonic solution by activating a Cl- current that was modestly outwards rectifying and inactivated at positive potentials. This current is similar to the well-described swelling-activated current through volume-regulated anion channels (VRAC) in endothelial cells. However, its activation is slower, its inactivation faster and the current density lower than in cultured endothelial cells. It is concluded that the primary explant technique provides a reliable cell model for studying mouse vascular endothelial cell function.

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.