TRPM2 channels, activated by adenosine diphosphoribose and related molecules, are assembled as oligomers and most likely tetramers. However, the molecular determinants driving the subunit interaction and assembly of the TRPM2 channels are not well defined. Here we examined, using site-directed mutagenesis in conjunction with co-immunoprecipitation and patch clamp recording, the role of a coiled-coil domain in the intracellular C terminus of TRPM2 subunit in subunit interaction and channel assembly. Deletion of the coiled-coil domain resulted in severe disruption of the subunit interaction and substantial loss of the adenosine diphosphoribose-evoked channel currents. Individual or combined mutations to glutamine of the hydrophobic residues at positions a and d of the abcdef heptad repeat, key residues for protein-protein interaction, significantly reduced the subunit interaction and channel currents; the mutational effects on the subunit interaction and channel currents were clearly correlated. Furthermore, deletion of the coiled-coil domain in a pore mutant subunit abolished its dominant negative phenotypic functional suppression. These results provide strong evidence that the coiled-coil domain is critically engaged in the TRPM2 subunit interaction and such interaction is required for assembly of functional TRPM2 channel. The coiled-coil domain, which is highly conserved within the TRPM subfamily, may serve as a general structural element governing the assembly of TRPM channels. Mammalian transient receptor potential (TRP)3 proteins, homologues of the Drosophila TRP proteins (1), form a large group of cation channels that are activated via diverse mechanisms and serve numerous physiological functions. They are subdivided into TRPC, TRPV, TRPM, TRPP, TRPML, and TRPA subfamilies based on sequence relatedness (2-5). TRPM2 channel, the second member of the TRPM subfamily, is activated by adenosine diphosphoribose (ADPR) and related molecules and also by exposure to oxidative stress and to warm temperature (6 -11). High expression of the TRPM2 channels is well documented in excitable cells and in particular in the brain (12-14), although the physiological roles are largely elusive. TRPM2 shows widespread expression in non-excitable cells, where the TRPM2 channels constitute a calcium entry system (7,8,11,(15)(16)(17).All the TRP family proteins, including TRPM2, have a membrane topology similar to the voltage-gated potassium channels, calcium-activated channels, and cyclic nucleotide-gated channels; each subunit comprises six transmembrane segments (S1-S6), a pore loop between the S5 and S6, and intracellular N and C termini (2-4, 18). TRPM2 subunit contains an N-terminal calmodulin binding site that mediates functional regulation of the TRPM2 channel by calmodulin (19) and a TRP motif in the proximal C-terminal part. Unique to the TRPM2 subunit, the distal C-terminal part shares substantial homology to the NUDT9 proteins (thus termed NUDT9-H domain). Between the TRP motif and NUDT9-H domain is a region showing a st...

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca 2؉ and Mg 2؉ permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg 2؉ permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca 2؉ permeability along with concentration-dependent inhibition of Na ؉ -carrying currents by Ca 2؉ . Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca 2؉ -permeable TRPM channels, substantially increased Ca 2؉ permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca 2؉ permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.The melastatin subtype of transient receptor potential (TRPM) 6 ion channels is widely expressed in neuronal, cardiovascular, immune, and endothelial cells where they are engaged in diverse physiological and pathophysiological processes (1-7). TRPM2 channels are activated by adenosine diphosphoribose (ADPR) and also by oxidative stress and mediate immune function, insulin secretion, endothelial permeability, and cell death that are induced by oxidative stress (8 -14).All members of the transient receptor potential (TRP) channel superfamily, which includes TRPC, TRPV, TRPM, TRPP, TRPML, and TRPA subfamilies, have a basic architecture similar to voltage-gated potassium channels, with homo-or hetero-tetrameric arrangements around a central ion-conducting pore (1,7,15). Each subunit is considered to have intracellular N and C termini and six transmembrane segments (S1-S6) with a re-entrant pore loop connecting S5 and S6 (see Fig. 1A). Despite significant differences in the amino acid residue sequences of the pore loop among different TRP subfamilies, there are two stretches of amino acid residues that are thought to form the pore helix and the ion selectivity filter of these channels, respectively (15).TRPM channels, including TRPM2, show considerable permeability to Ca 2ϩ and other divalent cations, with the exception of TRPM4/5 channels that are selective for monovalent cations (8, 9, 13-18). The molecular basis for divalent cationic permeability of TRPM channels is not fully understood. Accumulating evidence supports an important role of the ion selectivity filter in Ca 2ϩ and Mg 2ϩ permeation of TRPV and TRPM channels (15, 1...

Transient receptor potential melastatin 2 (TRPM2) channel fulfills an important role in oxidative stress signaling in immune and other cells, to which local extracellular acidosis is known to occur under physiological or pathological conditions and impose significant effects on their functions. Here, we investigated whether the ADP-ribose-activated TRPM2 channel is a target for modulation by extracellular acidic pH by patch clamp recording of HEK293 cells expressing hTRPM2 channel. Induced whole cell or single channel currents were rapidly inhibited upon subsequent exposure to acidic pH. The inhibition in the steady state was complete, voltage-independent, and pH-independent in the range of pH 4.0–6.0. The inhibition was irreversible upon returning to pH 7.3, suggesting channel inactivation. In contrast, exposure of closed channels to acidic pH reduced the subsequent channel activation in a pH-dependent manner with an IC50 for H+ of 20 μm (pH 4.7) and rendered subsequent current inhibition largely reversible, indicating differential or state-dependent inhibition and inactivation. Alanine substitution of residues in the outer vestibule of the pore including Lys952 and Asp1002 significantly slowed down or reduced acidic pH-induced inhibition and prevented inactivation. The results suggest that acidic pH acts as a negative feedback mechanism where protons bind to the outer vestibule of the TRPM2 channel pore and inhibit the TRPM2 channels in a state-dependent manner.

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.

We aimed to create a slippery liquid-infused enamel surface with antibiofouling property to prevent dental biofilm/plaque formation. First, a micro/nanoporous enamel surface was obtained by 37% phosphoric acid etching. The surface was then functionalized by hydrophobic low-surface energy heptadecafluoro-1,1,2,2-tetra-hydrodecyltrichlorosilane. Subsequent infusion of fluorocarbon lubricants (Fluorinert FC-70) into the polyfluoroalkyl-silanized rough surface resulted in an enamel surface with slippery liquid-infused porous surface (SLIPS). The results of water contact angle measurement, diffuse-reflectance Fourier transform infrared spectroscopy, and atomic force microscope confirmed that the SLIPS was successfully constructed on the enamel surface. The antibiofouling property of the SLIPS was evaluated by the adsorption of salivary protein of mucin and Streptococcus mutans in vitro, as well as dental biofilm formation using a rabbit model in vivo. The results showed that the SLIPS on the enamel surface significantly inhibited mucin adhesion and S. mutans biofilm formation in vitro, and inhibited dental plaque formation in vivo.