TRPM2 causes sensitization to oxidative stress but attenuates high-temperature injury in the sea anemone Nematostella vectensis.

In humans, the cation channel TRPM2 (HsTRPM2) has been intensively studied because it is involved in oxidative stress-mediated apoptosis and also contributes to temperature regulation. The gating mechanism of TRPM2 is quite complex, with a C-terminally localized enzyme domain playing a crucial role. The analysis of orthologues of TRPM2, in particular from the distantly related marine invertebrate Nematostella vectensis (NvTRPM2), revealed that during evolution, the functional role of the endogenous enzyme domain of TRPM2 has undergone fundamental changes. In this study, we investigated whether these evolutionary differences also apply to the physiological functions of TRPM2. For this purpose, we generated a TRPM2 loss-of-function mutation in N. vectensis and compared the phenotypes of wild-type and mutant animals after exposure to either oxidative stress or high temperature. Our results show that under standard culture conditions, mutant animals are indistinguishable from wild-type animals in terms of morphology and development. However, exposure of the two experimental groups to different stressors revealed that TRPM2 causes sensitization to oxidative stress but attenuates high-temperature injury in N. vectensis. Therefore, NvTRPM2 plays opposite roles in the cellular response to these two different stressors. These findings reveal a similar physiological spectrum of activity of TRPM2 in humans and N. vectensis and open up the possibility of establishing N. vectensis as a model organism for the physiological function of TRPM2.

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.

Structure-Function Relationship of TRPM2: Recent Advances, Contradictions, and Open Questions.

When in a particular scientific field, major progress is rapidly reached after a long period of relative stand-still, this is often achieved by the development or exploitation of new techniques and methods. A striking example is the new insights brought into the understanding of the gating mechanism of the transient receptor potential melastatin type 2 cation channel (TRPM2) by cryogenic electron microscopy structure analysis. When conventional methods are complemented by new ones, it is quite natural that established researchers are not fully familiar with the possibilities and limitations of the new method. On the other hand, newcomers may need some assistance in perceiving the previous knowledge in detail; they may not realize that some of their interpretations are at odds with previous results and need refinement. This may in turn trigger further studies with new and promising perspectives, combining the promises of several methodological approaches. With this review, I aim to give a comprehensive overview on functional data of several orthologous of TRPM2 that are nicely explained by structural studies. Moreover, I wish to point out some functional contradictions raised by the structural data. Finally, some open questions and some lines of possible future experimental approaches shall be discussed.

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.

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.

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.

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.

The transmembrane segment S6 determines cation versus anion selectivity of TRPM2 and TRPM8.

TRPM2 and TRPM8, closely related members of the transient receptor potential (TRP) family, are cation channels activated by quite different mechanisms. Their transmembrane segments S5 and S6 are highly conserved. To identify common structures in S5 and S6 that govern interaction with the pore, we created a chimera in which the S5-pore-S6 region of TRPM8 was inserted into TRPM2, along with a lysine at each transition site. Currents through this chimera were induced by ADP-ribose (ADPR) in cooperation with Ca(2+). In contrast to wild-type TRPM2 channels, currents through the chimera were carried by Cl(-), as demonstrated in ion substitution experiments using the cation N-methyl-D-glucamine (NMDG) and the anion glutamate. Extracellular NMDG had no effects. The substitution of either intracellular or extracellular Cl(-) with glutamate shifted the reversal potential, decreased the current amplitude and induced a voltage-dependent block relieved by depolarization. The lysine in S6 was responsible for the anion selectivity; insertion of a lysine into corresponding sites within S6 of either TRPM2 or TRPM8 created anion channels that were activated by ADPR (TRPM2 I1045K) or by cold temperatures (TRPM8 V976K). The positive charge of the lysine was decisive for the glutamate block because the mutant TRPM2 I1045H displayed cation currents that were blocked at acidic but not alkaline intracellular pH values. We conclude that the distal part of S6 is crucial for the discrimination of charge. Because of the high homology of S6 in the whole TRP family, this new role of S6 may apply to further TRP channels.

TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADP-ribose.

A unique functional property within the transient receptor potential (TRP) family of cation channels is the gating of TRP (melastatin) 2 (TRPM2) channels by ADP-ribose (ADPR). ADPR binds to the intracellular C-terminal tail of TRPM2, a domain that shows homology to enzymes with pyrophosphatase activity. Cytosolic Ca(2+) enhances TRPM2 gating by ADPR; ADPR and Ca(2+) in concert may be an important messenger system mediating Ca(2+) influx. Other stimuli of TRPM2 include NAD and H(2)O(2) and cyclic ADPR, which may act synergistically with ADPR. H(2)O(2), an experimental paradigm of oxidative stress, may also induce the formation of ADPR in the nucleus or mitochondria. In this review, we summarize the gating properties of TRPM2 and the proposed pathways of channel activation in vivo. TRPM2 is likely to be a key player in several signalling pathways, mediating cell death in response to oxidative stress or in reperfusion injury. Moreover, it plays a decisive role in experimentally induced diabetes mellitus and in the activation of leukocytes.

Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2.

TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.

Molecular cloning and functional characterization of a unique mammalian cardiac Na(v) channel isoform with low sensitivity to the synthetic inactivation inhibitor (-)-(S)-6-amino-alpha-[(4-diphenylmethyl-1-piperazinyl)-methyl]-9H-purine-9-ethanol (SDZ 211-939).

Cardiac voltage-dependent sodium channels (Na(v)) are drug targets for synthetic inactivation inhibitors typified by (+/-)-4- [3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxy propoxy]-1H-indole-2-carbonitrile (DPI 201-106), of which the molecular mode of action is not yet defined. The previous observation by Mevissen and coworkers in 2001 of the electrophysiological ineffectiveness of DPI 201-106 in the bovine heart, in contrast to other species, offers the opportunity for investigating these open questions. We now report about the molecular cloning, expression in Xenopus laevis oocytes, and electrophysiological characterization of a unique bovine heart sodium channel. Although the predicted 2022-amino acid bovine heart sodium channel (bH1) shares 92% identity with the rat and human isoforms and normal gating properties, it displays drastically reduced sensitivity to (-)-(S)-6-amino-alpha-[(4-diphenylmethyl-1-piperazinyl)-methyl]-9H-purine-9-ethanol (SDZ 211-939). Experimental results with Anemonia sulcata toxin II (0.1-2.5 microM) exclude the possibility of an overall insensitivity of this isoform to various sodium channel modulators. The binding of SDZ 211-939 seems to be largely unaffected (EC(50) of 10.3 and 10.6 microM for bovine and rat isoforms, respectively) but the corresponding efficacy in bovine (V(m) of 0.15) is approximately 5 times smaller compared with the rat heart isoform (V(m) of 0.69). The comparison of the primary structure of bH1 to other sodium channels and the gating properties obtained in presence or absence of SDZ 211-939 revealed a high degree of similarity. Whether the mechanism of channel modulation depends on the interaction of synthetic modulators with some possibly voltage-independent part of the inactivation machinery needs to be determined.