Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity.

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

Functional importance of NUDT9H domain and N-terminal ADPR-binding pocket in two species variants of vertebrate TRPM2 channels.

There are at least two different principles of how ADP-ribose (ADPR) induces activation of TRPM2 channels. In human TRPM2, gating requires the C-terminal NUDT9H domain as ADPR-binding module, whereas in sea anemone, NUDT9H is dispensable and binding of ADPR occurs N-terminally. Zebrafish TRPM2 needs both, the N-terminal ADPR-binding pocket and NUDT9H. Our aim was to pinpoint the relative functional contributions of NUDT9H and the N-terminal ADPR-binding pocket in zebrafish TRPM2, to identify fundamental mechanisms of ADPR-directed gating. We show that the NUDT9H domains of human and zebrafish TRPM2 are interchangeable since chimeras generate ADPR-sensitive channels. A point mutation at a highly conserved position within NUDT9H induces loss-of-function in both vertebrate channels. The substrate specificity of zebrafish TRPM2 corresponds to that of sea anemone TRPM2, indicating gating by the proposed N-terminal ADPR-binding pocket. However, a point mutation in this region abolishes ADPR activation also in human TRPM2. These findings provide functional evidence for an uniform N-terminal ADPR-binding pocket in TRPM2 of zebrafish and sea anemone with modified function in human TRPM2. The structural importance of NUDT9H in vertebrate TRPM2 can be associated with a single amino acid residue which is not directly involved in the binding of ADPR.

Different substrate specificities of the two ADPR binding sites in TRPM2 channels of Nematostella vectensis and the role of IDPR.

NvTRPM2 (Nematostella vectensis Transient Receptor Potential Melastatin 2), the species variant of the human apoptosis-related cation channel hTRPM2, is gated by ADP-ribose (ADPR) independently of the C-terminal NUDT9H domain that mediates ADPR-directed gating in hTRPM2. The decisive binding site in NvTRPM2 is likely to be identical with the N-terminal ADPR binding pocket in zebra fish DrTRPM2. Our aim was a characterization of this binding site in NvTRPM2 with respect to its substrate specificity, in comparison to the classical ADPR interaction site within NUDT9H that is highly homologous in hTRPM2 and NvTRPM2, although only in NvTRPM2, catalytic (ADPRase) activity is conserved. With various ADPR analogues, key differences of the two sites were identified. Particularly, two reported antagonists on hTRPM2 were agonists on NvTRPM2. Moreover, IDP-ribose (IDPR) induced currents both in hTRPM2 and NvTRPM2 but not in NvTRPM2 mutants in which NUDT9H was absent. Thus, IDPR acts on NUDT9H rather than N-terminally, revealing a regulatory function of NUDT9H in NvTRPM2 opposed to that in hTRPM2. We propose that IDPR competitively inhibits the ADPRase function of NUDT9H and evokes ADPR accumulation. The findings provide important insights into the structure-function relationship of NvTRPM2 and will allow further characterization of the novel ADPR interaction site.

Different Principles of ADP-Ribose-Mediated Activation and Opposite Roles of the NUDT9 Homology Domain in the TRPM2 Orthologs of Man and Sea Anemone.

A decisive element in the human cation channel TRPM2 is a region in its cytosolic C-terminus named NUDT9H because of its homology to the NUDT9 enzyme, a pyrophosphatase degrading ADP-ribose (ADPR). In hTRPM2, however, the NUDT9H domain has lost its enzymatic activity but serves as a binding domain for ADPR. As consequence of binding, gating of the channel is initiated. Since ADPR is produced after oxidative DNA damage, hTRPM2 mediates Ca2+ influx in response to oxidative stress which may lead to cell death. In the genome of the sea anemone Nematostella vectensis (nv), a preferred model organism for the evolution of key bilaterian features, a TRPM2 ortholog has been identified that contains a NUDT9H domain as well. Heterologous expression of nvTRPM2 in HEK-293 cells reveals a cation channel with many close similarities to the human counterpart. Most notably, nvTRPM2 is activated by ADPR, and Ca2+ is a co-agonist. However, the intramolecular mechanisms of ADPR gating as well as the role of NUDT9H are strikingly different in the two species. Whereas already subtle changes of NUDT9H abolish ADPR gating in hTRPM2, the region can be completely removed from nvTRPM2 without loss of responses to ADPR. An alternative ADPR binding site seems to be present but has not yet been characterized. The ADP-ribose pyrophosphatase (ADPRase) function of nvNUDT9H has been preserved but can be abolished by numerous genetic manipulations. All these manipulations create channels that are sensitive to hydrogen peroxide which fails to induce channel activity in wild-type nvTRPM2. Therefore, the function of NUDT9H in nvTRPM2 is the degradation of ADPR, thereby reducing agonist concentration in the presence of oxidative stress. Thus, the two TRPM2 orthologs have evolved divergently but nevertheless gained analogous functional properties, i.e., gating by ADPR with Ca2+ as co-factor. Opposite roles are played by the respective NUDT9H domains, either binding of ADPR and mediating channel activity, or controlling the availability of ADPR at the binding site located in a different domain.

Modulation of activation and inactivation by Ca2+ and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis.

The archetypal TRPM2-like channel of the sea anemone Nematostella vectensis is gated by ADPR like its human orthologue but additionally exhibits properties of other vertebrate TRPM channels. Thus it can help towards an understanding of gating and regulation of the whole subfamily. To elucidate further the role of Ca2+ as a co-factor of ADPR, we exploited 2-aminoethyl diphenylborinate (2-APB), previously shown to exert either inhibitory or stimulatory effects on diverse TRPM channels, or both in a concentration-dependent manner. 2-APB in high concentrations (1 mM) induced large, non-inactivating currents through nvTRPM2. In lower concentrations (≤0.5 mM), it prevented the fast current inactivation typical for nvTRPM2 stimulated with ADPR. Both these effects were rapidly reversed after wash-out of 2-APB, in contrast to a considerable lag time of their onset. A detailed analysis of nvTRPM2 mutants with modified selectivity filter or reduced ADP-ribose sensitivity revealed that the actions of 2-APB depend on its access to the pore which is enhanced by channel opening. Moreover, access of Ca2+ to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca2+ on the pore can be discriminated with the aid of 2-APB.

ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain.

The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of hTRPM2. The TRPM2 orthologue from Nematostella vectensis (nv) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca2+-imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells. We demonstrate that the removal of the entire NUDT9H domain does not disturb the response of nvTRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nvTRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nvTRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original hNUDT9 enzyme. Whenever the enzyme domain was mutated to presumably inactive variants, channel activation by hydrogen peroxide could be achieved. Moreover, we found strong evidences for ADPRase activity of the isolated NUDT9H domain of nvTRPM2 in co-expression experiments with the C-terminally truncated nvTRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nvTRPM2 to respond to oxidative stress. In striking contrast, the channel function of the hTRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nvTRPM2 as a channel gated by ADP-ribose through a novel mechanism. We conclude that the endogenous NUDT9H domain does not directly affect ADP-ribose-dependent gating of the nvTRPM2 channel; instead it exerts an independent catalytic function which possibly controls the intracellular availability of ADP-ribose.

Functional characterisation of a TRPM2 orthologue from the sea anemone Nematostella vectensis in human cells.

The human non-selective cation channel TRPM2 represents a mediator of apoptosis triggered by oxidative stress. The principal agonist ADP-ribose binds to the cytosolic domain of TRPM2, which is homologous to the human ADP-ribose pyrophosphatase NUDT9. To further elucidate the structure-function relationship of this channel, we characterised a TRPM2 orthologue from the cnidarian Nematostella vectensis, after its expression in a human cell line. This far distant relative shows only 31% total sequence similarity to hTRPM2, while its C-terminal domain has a greater resemblance to the NUDT9 enzyme. Current through nvTRPM2 was induced by ADPR, with a more pronounced sensitivity and faster kinetics than in hTRPM2. In contrast to hTRPM2, there was no response to H2O2 and hardly any modulatory effect by intracellular Ca(2+). The deletion of a stretch of 15 residues from the NUDT9 domain of nvTRPM2, which is absent in hTRPM2, did not change the response to ADPR but enabled activation of the channel by H2O2 and increased the effects of intracellular Ca(2+). These findings shed new light on the evolution of TRPM2 and establish nvTRPM2 as a promising tool to decipher its complex gating mechanisms.

Surface expression and channel function of TRPM8 are cooperatively controlled by transmembrane segments S3 and S4.

TRPM8 is a voltage-dependent cation channel additionally gated by cold temperatures, menthol, and icilin. Stimulation by the chemical agonists is at least in part mediated by a conserved sequence motif in transmembrane segment S3. Based on molecular dynamics simulation studies for TRPM8 a gating model was recently developed which predicts a direct electrostatic interaction between S3 and S4. Here, we performed charge reversal mutations to pinpoint possible interactions of the putative S4 voltage sensor with S3. The charge reversals R842D, R842E, and D835R in S4 prevented channel glycosylation and function, indicating a deficient insertion into the plasma membrane. The mutations R842D and R842E were specifically rescued by the reciprocal charge reversal D802R in S3. The alternative charge reversal in S3, D796R, failed to compensate for the dysfunction of the mutants R842D and R842E. Remarkably, the double charge reversal mutants R842D + D802R and R842E + D802R retained intrinsic voltage-sensitivity, although the critical voltage sensor arginine was substituted by a negatively charged residue. Likewise, the insertion of three additional positively charged residues into S4 did not crucially change the voltage-sensitivity of TRPM8 but abolished the sensitivity to icilin. We conclude that S4 does not play a separate role for the gating of TRPM8. Instead, the cooperation with the adjacent segment S3 and the combined charges in these two segments is of general importance for both channel maturation and channel function. This mechanism distinguishes TRPM8 from other voltage-dependent cation channels within and outside the TRP family.

Importance of a conserved sequence motif in transmembrane segment S3 for the gating of human TRPM8 and TRPM2.

For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating.

Comparing ion conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels.

In this article we compare electrical conductance events from single channel recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in human embryonic kidney cells with channel events recorded on synthetic lipid membranes close to melting transitions. Ion channels from the TRP family are involved in a variety of sensory processes including thermo- and mechano-reception. Synthetic lipid membranes close to phase transitions display channel-like events that respond to stimuli related to changes in intensive thermodynamic variables such as pressure and temperature. TRP channel activity is characterized by typical patterns of current events dependent on the type of protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical phenomena that are considered typical for the activity of protein ion channels. We find unitary currents, burst behavior, flickering, multistep-conductances, and spikes behavior in both preparations. Moreover, we report conductances and lifetimes for lipid channels as described for protein channels. Non-linear and asymmetric current-voltage relationships are seen in both systems. Without further knowledge of the recording conditions, no easy decision can be made whether short current traces originate from a channel protein or from a pure lipid membrane.

Impaired somatosensation in tongue mucosa of smokers.

Smoking has been indicated as a risk factor for oral diseases and can lead to altered sense of taste. So far, the effects of sensory changes on the tongue are not investigated. In this study, quantitative sensory testing was used to evaluate somatosensory function in the lingual region. Eighty healthy volunteers were investigated (20 smokers, 20 non-smokers). Subjects were bilaterally tested in innervation areas of lingual nerves. Thresholds of cold and warm detection, cold and heat pain, and mechanical detection were determined. As control for systemic, extraoral effects of smoking, tests were additionally performed in 40 volunteers (20 smokers, 20 non-smokers) on the skin of the chin innervated by the mental branch of the trigeminal nerve. Cold (p < 0.001), warm detection thresholds (p < 0.001), and thermal sensory limen (p < 0.001) showed higher sensitivity in non-smokers as compared to smokers. Heat pain and mechanical detection, as well as all tests in the skin of the chin, showed no significant differences. The impaired temperature perception in smokers indicates a reduction of somatosensory functions in the tongue, possibly caused by nerve degeneration associated with smoking. Possible systemic effects of smoking do not seem to affect extraoral trigeminal branches.

Contribution of the S5-pore-S6 domain to the gating characteristics of the cation channels TRPM2 and TRPM8.

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

Inhibitors of TRP channels reveal stimulus-dependent differential activation of Ca2+ influx pathways in human neutrophil granulocytes.

A pharmacological characterization of Ca(2+) influx pathways in neutrophil granulocytes is problematic because of the lack of specific inhibitors. The activation of transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channels by intracellular adenosine diphosphate ribose (ADPR), well characterized in neutrophils, is reportedly inhibited by 8-bromo-ADPR (8Br-ADPR). TRPM2 is blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA) interfering with the pore, but ACA is as well effective on other transient receptor potential channels, especially transient receptor potential canonical (TRPC) channels. We wished to analyze whether ACA and 8Br-ADPR were suitable probes to demonstrate that different Ca(2+) entry pathways are activated in human neutrophil granulocytes by the receptor-dependent stimuli N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and platelet-activating factor (PAF) and the receptor-independent thapsigargin. Ca(2+)-influx-related increases in [Ca(2+)](i) were calculated by comparing aliquots of fluo-3-loaded neutrophils in the presence and absence of extracellular Ca(2+). Moreover, Mn(2+) quenching was used in fura-2-loaded cells. We compared 8Br-ADPR with ACA. 8Br-ADPR was exclusively effective when Ca(2+) influx (or Mn(2+) quenching) was induced by fMLP; it did not affect influx when PAF or thapsigargin was the stimulus. ACA inhibited Ca(2+) influx significantly more strongly when this was induced by PAF than by fMLP. Moreover, it reduced thapsigargin-induced Ca(2+) influx. The contribution of TRPM2 to Ca(2+) influx in neutrophils strongly depends on the stimulus; it is sizeable in the case of fMLP and minimal in the case of PAF. PAF induces Ca(2+) entry pathways different from TRPM2; the inhibition by ACA suggests the contribution of channels of the TRPC family.

Pharmacological modulation of transmitter release by inhibition of pressure-dependent potassium currents in vestibular hair cells.

Vestibular vertigo may be induced by increases in the endolymphatic pressure that activate pressure-dependent K(+) currents (I(K,p)) in vestibular hair cells. I(K,p) have been demonstrated to modulate transmitter release and are inhibited by low concentrations of cinnarizine. Beneficial effects against vestibular vertigo of cinnarizine have been attributed to its inhibition of calcium currents. Our aim was to determine the extent by which the inhibition of I(K,p) by cinnarizine may alter the voltage response to stimulating currents and to analyze whether such alterations may be sufficient to modulate the activation of Ca(2+) currents and transmitter release. Vestibular type II hair cells from guinea pigs were studied using the whole-cell patch-clamp technique. In current clamp, voltage responses to trains of stimulating currents were recorded. In voltage clamp, transmitter release was assessed from changes in the cell capacitance, as calculated from the phase shift during application of sine waves. Cinnarizine (0.05-3 microM) concentration dependently reversed the depressing effects of increases in the hydrostatic pressure (from 0.2 to 0.5 cm H(2)O) on the voltage responses to stimulating currents. Voltage protocols that simulated these responses were applied in voltage clamp and revealed a significantly enhanced transmitter release in conditions mimicking an inhibition of I(K,p). Cinnarizine (< or =0.5 microM) did not inhibit calcium currents. We conclude that cinnarizine, in pharmacologically relevant concentrations, enhances transmitter release in the presence of elevated hydrostatic pressure by an indirect mechanism, involving inhibition of I(K,p), enhancing depolarization, and increasing the voltage-dependent activation of Ca(2+) currents, without directly affecting Ca(2+) current.

H2O2-induced Ca2+ influx and its inhibition by N-(p-amylcinnamoyl) anthranilic acid in the beta-cells: involvement of TRPM2 channels.

Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca(2+)-permeable channel activated by hydrogen peroxide (H(2)O(2)). We have investigated the role of TRPM2 channels in mediating the H(2)O(2)-induced increase in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of beta-cells, and in human beta-cells, H(2)O(2) increased [Ca(2+)](i), in the presence of 3 mM glucose, by inducing Ca(2+) influx across the plasma membrane. H(2)O(2)-induced Ca(2+) influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca(2+) channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H(2)O(2), induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H(2)O(2)-induced Ca(2+) entry in beta-cells, a process potently inhibited by ACA.

Inhibition of TRPM8 by icilin distinct from desensitization induced by menthol and menthol derivatives.

TRPM8 is a cation channel activated by cold temperatures and the chemical stimuli menthol and icilin. Both compounds use different mechanisms of current activation; amino acid residues within the S2-S3 linker have been identified critical for current activation by icilin but not by menthol. Current decline in the course of menthol stimulation reflects Ca(2+)-dependent desensitization attributed to phosphatidylinositol 4,5-bisphosphate depletion. Carboxyamide derivatives chemically resembling menthol have been described as activators of TRPM8 analogous to icilin. Our aim was a detailed analysis of whether differences exist between all these substances with respect to their activation and inactivation of currents. We studied wild-type TRPM8 as well as an s3-TRPM8 mutant with mutations in the S2-S3 linker region that could not be activated by icilin. Menthol and menthol derivatives behaved indistinguishable in evoking currents through both channels in a Ca(2+)-independent manner as well as inducing Ca(2+)-dependent desensitization. Icilin, in contrast, activated currents only in wild type TRPM8 and in the presence of Ca(2+). Moreover, it completely reversed currents induced by menthol, menthol derivatives, and cold temperatures in wild type TRPM8 and s3-TRPM8; this current inhibition was independent of Ca(2+). Finally, icilin suppressed current activation by the other agonists. None of the inhibiting effects of icilin occurred in the cation channel TRPA1 that is also stimulated by both menthol and icilin. Thus, icilin specifically inhibits TRPM8 independently of its interaction site within the S2-S3 linker through a process distinct from desensitization.

Role of an N-terminal splice segment in the activation of the cation channel TRPM2 by ADP-ribose and hydrogen peroxide.

In the dysfunctional splice variant TRPM2-DeltaN, a stretch of 20 amino acids (aa 537-556) is missing within the N-terminal cytosolic tail of the cation channel TRPM2. The DeltaN-stretch overlaps with two IQ-like calmodulin-binding domains. Moreover, it contains two PxxP motifs implicated in protein-protein interactions. Here, we constructed variants to test whether any of these motifs may explain why TRPM2-DeltaN does not respond to stimulation with either ADP ribose or hydrogen peroxide. Each of the two IQ-motifs could be removed without loss of channel function. Similarly, deletion of either one or both PxxP motifs had no effect. Moreover, the single point mutation D543E associated with bipolar disorder does not change the activation of TRPM2. We conclude that no functional role can be attributed to any of the structural motifs within the DeltaN-stretch that may be a spacer segment for other functional sites in the N terminus.

Potassium currents induced by hydrostatic pressure modulate membrane potential and transmitter release in vestibular type II hair cells.

Vestibular type II hair cells respond to increases in the hydrostatic pressure with pressure-dependent K(+) currents. We examined whether such currents may modulate transmitter release (assessed as membrane capacitance increments) by altering membrane potentials and voltage-gated Ca(2+) currents. Capacitance increments were dependent on voltage-gated Ca(2+) influx. Stimulating currents (0.7 nA) in current clamp induced depolarisations that were more negative by 8.7 +/- 2.1 mV when the bath height was elevated from 0.2 to 0.5 cm. In voltage clamp, protocols were used that simulated the time course of the membrane potential in current clamp at either low (control) or high hydrostatic pressure (high bath). The low bath protocol induced significantly larger Ca(2+) currents and increases in capacitance than the high bath protocol. We conclude that pressure-dependent K(+) currents may alter the voltage response of vestibular hair cells to an extent critical for Ca(2+) currents and transmitter release. This mechanism may contribute to vestibular dysfunction in Meniere's disease.

Effects of antioxidants on calcium influx through TRPM2 channels in transfected cells activated by hydrogen peroxide.

Melastatin-like transient receptor potential 2 (TRPM2) channel is a redox sensitive Ca(2+)-permeable cation channel that can be gated by H(2)O(2) binding to the channel's enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 action in response to H(2)O(2) are not understood, we examined the effects of various antioxidants on H(2)O(2)-induced TRPM2 cation channel currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. Membrane currents were measured with the conventional whole cell patch-clamp technique. The intracellular solution contained ethylenediamine tetraacetic acid (EDTA) as a chelator for Ca(2+) and heavy metal ions instead of ethylene glycol tetraacetic acid (EGTA). Moreover, we chose an intracellular Ca(2+) concentration calculated to be in the range of 1 microM. H(2)O(2) (10 mM) was added extracellularly to the bath chamber. With these conditions, we were able to evoke TRPM2 currents consistently with H(2)O(2). We next tested whether vitamins C and E or glutathione (GSH) would prevent or attenuate the induction of TRPM2 currents by H(2)O(2) when applied extracellularly or intracellularly. Unexpectedly, administration of these antioxidants did not inhibit activation of TRPM2 by H(2)O(2). In conclusion, TRPM2 channels were constitutively activated by H(2)O(2) although we could not detect any inhibitory effect of the antioxidants on H(2)O(2)-induced TRPM2 cation channel currents in CHO cells.

A calcium influx pathway regulated separately by oxidative stress and ADP-Ribose in TRPM2 channels: single channel events.

A melastatin-like transient receptor potential 2 (TRPM2) channel is activated in concert with Ca2+ by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzyme Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2) although the mechanisms remain unknown. Hence, we tested the effects of ADPR, NAD+ and H2O2 on the activation of TRPM2 currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. The intracellular solution used EDTA (10 mM) as a chelator for Ca2+ and heavy metal ions. Moreover, we balanced the intracellular Ca2+ concentration at 1 microM. H2O2 (10 mM) in the bath chamber was extracellularly added although ADPR (0.3 mM) and NAD+ (1 mM) in pipette solution were intracellularly added. Using these conditions, the channel currents were evoked by the three stimulators. The time course of ADPR, NAD+ and H2O2 effects was characterized by a delay of 0.6, 3.0 min and 2-5 min, respectively and a slow current induction reached a clear plateau with ADPR and NAD+ although H2O2 currents continued to gain in amplitude over at least 15 min and it did not reach a clear plateau in many experiments. Furthermore, H2O2-induced a single-channel conductance in the current study; the first time that this has been resolved in CHO. The conductance of ADPR and H2O2 was 48.80 pS and 39.14 pS, respectively and the cells seem to be separately activated by ADPR and H2O2. In conclusion, we observed further support for a calcium influx pathway regulated separately by oxidative stress and ADPR in TRPM2 channels in transfected cells. A second novel result of the present study was that the TRPM2 channels were constitutionally activated by H2O2.