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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

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