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TRPM2 is a [Ca.sup.2+]-permeable nonselective cation channel that plays important roles in oxidative stress-mediated cell death and inflammation processes. However, how TRPM2 is regulated under physiological and pathological conditions is not fully understood. Here, we report that both intracellular and extracellular protons block TRPM2 by inhibiting channel gating. We demonstrate that external protons block TRPM2 with an [IC.sub.50] of p[H.sub.o] = 5.3, whereas internal protons inhibit TRPM2 with an [IC.sub.50] of p[H.sub.i] = 6.7. Extracellular protons inhibit TRPM2 by decreasing single-channel conductance. We identify three titratable residues, H958, D964, and E994, at the outer vestibule of the channel pore that are responsible for p[H.sub.o] sensitivity. Mutations of these residues reduce single-channel conductance, decrease external [Ca.sup.2+] ([[[Ca.sup.2+]].sub.o]) affinity, and inhibit [[[Ca.sup.2+]].sub.o]-mediated TRPM2 gating. These results support the following model: titration of H958, D964, and E994 by external protons inhibits TRPM2 gating by causing conformation change of the channel, and/or by decreasing local [Ca.sup.2+] concentration at the outer vestibule, therefore reducing [[[Ca.sup.2+]].sub.o] permeation and inhibiting [[[Ca.sup.2+]].sub.o]-mediated TRPM2 gating. We find that intracellular protons inhibit TRPM2 by inducing channel closure without changing channel conductance. We identify that D933 located at the C terminus of the $4-$5 linker is responsible for intracellular pH sensitivity. Replacement of [Asp.sup.933] by [Asn.sup.933] changes the [IC.sub.50] from p[H.sub.i] = 6.7 to p[H.sub.i] = 5.5. Moreover, substitution of [Asp.sup.933] with various residues produces marked changes in proton sensitivity, intracellular ADP ribose/[Ca.sup.2+] sensitivity, and gating profiles of TRPM2. These results indicate that D933 is not only essential for intracellular pH sensitivity, but it is also crucial for TRPM2 channel gating. Collectively, our findings provide a novel mechanism for TRPM2 modulation as well as molecular determinants for pH regulation of TRPM2. Inhibition of TRPM2 by acidic pH may represent an endogenous mechanism governing TRPM2 gating and its physiological/pathological functions. doi/ 10.1085/jgp.200910254

Melastatin-related transient receptor potential channel 2 (TRPM2) is a [Ca.sup.2+]-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [[[Ca.sup.2+]].sub.i], indicating that [[[Ca.sup.2+]].sub.i] gating of TRPM2 is independent of ADPR. The mechanism by which [[[Ca.sup.2+]].sub.i] activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas [Ca.sup.2+]-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [[[Ca.sup.2+]].sub.i] and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular [Ca.sup.2+] release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced [Ca.sup.2+] release is capable of activating TRPM2. These results indicate that [[[Ca.sup.2+]].sub.i] is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [[[Ca.sup.2+]].sub.i]-mediated activation of TRPM2 and its alternative spliced isoforms may representa major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [[[Ca.sup.2+]].sub.i]. [Ca.sup.2+] signaling | gating mechanism | ADP-ribose | calmodulin-binding domain | oxidative stress

TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including [Mg.sup.2+] homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM[6.sup.S141L] produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, [Mg.sup.2+] and [Ca.sup.2+] entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo.

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at + 100 mV but a small inward current at voltages ranging from--100 to -40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an ~10-fold increase at pH 4.0 and 1-2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with [Ca.sup.2+] and [Mg.sup.2+] for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external [Ca.sup.2+] and [Mg.sup.2+ concentrations were increased. Third, the apparent] affinity for [Ca.sup.2+] and [Mg.sup.2+] was significantly diminished at elevated external [H.sup.+] concentrations. Fourth, the anomalous-mole fraction behavior of [H.sup.+] permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), [Ca.sup.2+] and [Mg.sup.2+] bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high [H.sup.+] concentrations, the affinity of TRPM7 for [Ca.sup.2+] and [Mg.sup.2+] is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as [Mg.sup.2+]-inhibitable cation current (MIC) or Mg nucleotide--regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 200lb. Science. 294:2372-2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated [Ca.sup.2+] (Cav) and [Na.sub.+] (Nav) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the [Na.sub.+]-selective NaChBac to a [Ca.sup.2+]- and [Na.sub.+]-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated [Ca.sup.2+]-selective conductance. KEY WORDS: bacterial channels * calcium channels * sodium channels * ion/membrane channel

The pore-forming subunits of canonical voltage-gated sodium and calcium channels are encoded by four repeated domains of six-transmembrane (6TM) segments. We expressed and characterized a bacteria[ ion channel (NaChBac) from Bacillus halodurans that is encoded by one 6TM segment. The sequence, especially in the pore region, is similar to that of voltage-gated calcium channels. The expressed channel was activated by voltage and was blocked by calcium channel blockers. However, the channel was selective for sodium. The identification of NaChBac as a functionally expressed bacterial voltage-sensitive ion-selective channel provides insight into both voltage-dependent activation and divalent cation selectivity., Voltage-gated potassium ([K.sub.v]), sodium ([Na.sub.v]), and calcium ([Ca.sub.v]) channels underpin specialized higher order cell functions such as excitability, contraction, secretion, and synaptic transmission (1). Hundreds of [K.sub.v], [Na.sub.v], and [Ca.sub.v] [...]

Author(s): Lixia Yue [1]; Ji-Bin Peng [2]; Matthias A. Hediger [2]; David E. Clapham (corresponding author) [1, 3] The calcium-release-activated Ca[sup.2+] channel, I [sub.CRAC] [1, 2, 3], is a highly [...]

Research was conducted to examine the transmural heterogeneity of action potentials and I(sub to1) in myocytes isolated from the human right ventricle. Results reveal that M cells are present in the right ventricle in humans and that a gradient of I(sub to1) is present in cells across the human right ventricular wall from the endocardium to the epicardium. Findings show important transmural heterogeneity in the action potential and I(sub to1) properties pointing to an important clinical implication in human cardiac electrophysiology.

Isolation of cells by chunk and perfusion methods reveals that the transient outward K+ current, (Ito1), Ca2+ dependent Cl- current, (Ito2), and the delayed rectifier K+ current, (Ik), are found in the dog atrium. Ik is more sensitive to the isolation method than Ito1 or Ito2. The properties of Ito1 and Ik repolarizing currents of the dog atrium are similar to those in the human atrium. These studies help in the in vivo and in vitro analysis of canine atrial electrophysiology.

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing [Alpha]-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function., Phototransduction in Drosophila invokes phospholipase C (PLC)-mediated activation of transient receptor potential (TRP) channels, leading to membrane depolarization (1, 2). The mammalian TRP channel family may be divided by sequence [...]