Ca2+ mobilization from intracellular stores represents an important cell signaling process 1 which is regulated, in mammalian cells, by inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cADPR release Ca2+ from sarco / endoplasmic reticulum (S/ER) stores through activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). By contrast, the nature of the intracellular stores targeted by NAADP and molecular identity of the NAADP receptors remain controversial 1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments 3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 and TPC3 being expressed on endosomal and TPC2 on lysosomal membranes. Membranes enriched with TPC2 exhibit high affinity NAADP binding and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release via InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but only attenuated by depleting ER Ca2+ stores or blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger additional Ca2+ signals via S/ER. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells and will advance our understanding of the physiological role of NAADP.

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP)1 superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1 and 2). These channels are involved in diverse cellular functions including receptor and store-operated Ca 2ϩ entry (3), Ca 2ϩ transport (4, 5), trace metal detection (6), and temperature (7-9) and osmolality (10, 11) sensations. The activation mechanisms for most of the TRP channels remain to be elucidated. Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12), anandamide (13, 14), and phosphatidylinositol 4,5-bisphosphate (15)), nucleotides (ADP-ribose (16) (23,24), 2APB was soon found to directly block native store-operated channels (25-27), sarco/ endoplasmic reticulum Ca 2ϩ -ATPase pumps (28), mitochondrial permeability transition pore (29), and a few other ion channels (30). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26). At greater than 50 M, 2APB activated a Ca 2ϩ -permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31).2APB has been perceived as a general inhibitor of TRP channels (1). However, except for TRPC3 (24,32), the effects of this drug...

TRPM4, a Ca2؉ -activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca 2؉ . The mechanisms underlying the alterations in Ca 2؉ sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca 2؉ 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 Ca 2؉ sensitivity of wildtype TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca 2؉ dramatically reduced TRPM4 activation. We identified five Ca 2؉ -calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca 2؉ sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca 2؉ sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus. TRPM41 is a Ca 2ϩ -activated and voltage-dependent Ca 2ϩ -impermeable cation channel with a unitary conductance of 25 picosiemens that belongs to the melastatin subfamily of transient receptor potential membrane proteins (1-4). Ca 2ϩ -activated, Ca 2ϩ -impermeable nonselective cation channels that share functional properties with expressed TRPM4 (or the closest homologue, TRPM5) have been found in many excitable and non-excitable cells (Refs. 5-8; for reviews, see Refs. 9 and 10). These nonselective channels may regulate important processes including cardiac rhythmicity and neural bursting activity, and their Ca 2ϩ -dependent activation has been suggested to form a general feedback control mechanism for Ca 2ϩ influx in nonexcitable cells.The functional analysis of TRPM4 and TRPM5 and their comparison with native nonselective cation channels are complicated by a peculiar property of these channels: when activated by an increase in free intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), the currents decay rapidly due to a decrease in the sensitivity of the channels to Ca 2ϩ (1,3,4,11). Moreover, recent studies on TRPM4 exhibit an unusually large variability in reported values for Ca 2ϩ sensitivity and activation time courses (1-4). Most likely, these discrepancies reflect a highly regulated Ca 2ϩ affinity of TRPM4, which may be of physiological relevance.Another puzzling property of TRPM4 is its sensitivity to ATP. We have shown recently that cytosolic ATP 4Ϫ acts as a potent inhibitor of TRPM4 currents in inside-out patches, with half-maximal inhibition at ϳ2 M (3). However, robust TRPM4 currents can be measured in the whole-cell mode, even under conditions in which the free cytosolic ATP 4Ϫ concentration exceeds 100 M. One possible explanation of this apparent paradox could be that ATP has both an inhibitory and a stimulatory effect on TRPM4, but experimental data to support this idea are currently lacking.In the present study, we investigated potential cellular...

permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity. TRPM41 is a Ca 2ϩ -and voltage-dependent non-selective cation channel belonging to the melastatin subfamily of transient receptor potential (TRP) membrane proteins (1, 2). It has been proposed to be the molecular correlate of Ca 2ϩ -activated nonselective cation channels in several excitable and non-excitable cell types, and it has been implicated in important physiological processes including T-cell activation, myogenic vasoconstriction, and cardiac function (3-5).TRPM4 and its close homologue, TRPM5, exhibit two salient features that are unique within the TRP superfamily. First, they represent the only known TRP channels that are directly gated by increases in intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (1, 2, 6 -11). The Ca 2ϩ sensitivity of TRPM4 activation is strongly modulated by several cellular factors, including protein kinase C phosphorylation, calmodulin binding, and ATP (11, 12). Second, both channels are impermeable to Ca 2ϩ (1, 6 -8). This contrasts with all other functionally expressed TRPs, which form either Ca 2ϩ -permeable non-selective cation channels or even highly Ca 2ϩ -selective channels. The structural basis of TRPM channel permeation has not yet been studied. The region between the fifth and sixth transmembrane helices (TM5 and TM6), which is known to form the pore in the other tetrameric cation channels, shows only limited sequence homology to other (TRP) cation channels but is highly conserved among members of the TRPM subfamily. This region consists of a conserved hydrophobic region, a putative pore helix, followed by a hydrophilic region that contains a fully conserved aspartate residue (residue 984 in TRPM4), which may be part of the selectivity filter (13). There are, however, some clear sequence differences. In TRPM4 and TRPM5, the putative selectivity filter is highly acidic, with a cluster of three (TRPM4) or four (TRPM5) aspartates or glutamates. The other TRPM channels, which all exhibit some degree of Ca 2ϩ permeability, have only one or two acidic residues in this region. Moreover, the span between the putative pore helix and selectivity filter is one amino acid shorter in TRPM2 and TRPM8 than in the other TRPM channels. We hypothesized that the lack of Ca 2ϩ permeability of the TRPM4 and TRPM5 pores may be related to these structural differences.In this study, we used a site-directed mutagenesis approach to investigate how changes to the putative selectivity filter affect the pore properties of TRPM4. Our data indicate that this region determines the TRPM4 permeability properties and its sensitivity to block by intracellular spermine. EXPERIMENTAL PROCEDURESCell Culture-HEK293 human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37°C in a humidity-controlled incubator with 10% CO 2 .Transient ...

Transient receptor potential channels are involved in sensing chemical and physical changes inside and outside of cells. TRPV3 is highly expressed in skin keratinocytes, where it forms a nonselective cation channel activated by hot temperatures in the innocuous and noxious range. The channel has also been implicated in flavor sensation in oral and nasal cavities as well as being a molecular target of some allergens and skin sensitizers. TRPV3 is unique in that its activity is sensitized upon repetitive stimulations. -mediated inhibition and greatly facilitated the activation of TRPV3. We conclude that Ca 2؉ inhibits TRPV3 from both the extracellular and intracellular sides. The inhibition is sequentially reduced, appearing as sensitization to repetitive stimulations. Members of the transient receptor potential (TRP)4 superfamily of cation channels have been recognized to play important roles in sensing various environmental changes inside and outside of cells as well as the whole organisms (1). In mammals, temperature sensing is thought to be accomplished through concerted actions of a minimum of six TRP channels, i.e. TRPA1, -M8, -V4, -V3, -V1, and -V2, each covering a defined temperature range from below 17°C to above 52°C (2, 3). However, the debate remains whether some of these channels, e.g. TRPA1, are really temperature-sensitive (4). In addition, TRPM2, -M4, and -M5 have shown temperature sensing in the presence of second messenger cofactors, such as ADP-ribose and Ca 2ϩ (5, 6). Although some of the thermosensitive TRP channels are clearly expressed and functional in sensory neurons, indicative of their actions in primary afferents, others have been localized in the non-nervous tissues, for example, TRPV3 and -V4 are expressed in skin keratinocytes (7,8) and TRPV3 is, in addition, expressed in the epithelium of tongue and nose (9). The TRPV3 null mice showed some deficits in sensing hot temperatures in the innocuous and noxious range but no other obvious sensory impairment (10). On the other hand, constitutively active mutations of TRPV3 have been linked to hair loss and atopic dermatitis-like skin lesions in rodents (11,12).In addition to temperature, the thermosensitive TRP channels are activated by a large number of structurally unrelated chemical ligands from exogenous as well as endogenous sources (13). This polymodal nature has become a common feature of the TRP channel family, implicating that multiple mechanisms and external stimuli may be involved in the activation and regulation of these channels. TRPV3 was first shown to be activated by 2-aminoethoxydiphenyl borate (2APB), a synthetic compound known to inhibit inositol 1,4,5-trisphosphate receptors and store-operated channels as well as many TRP channels (14, 15). It was soon discovered that a number of natural anti-irritants and flavor enhancers such as camphor, carvacrol, thymol, and eugenol, also use TRPV3 as one of their targets (9, 10). More importantly, cell signaling events leading to the activation of phospholipase C, phosphorylation by...