The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzymatic Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2), but their mechanisms of action remain unknown. Here, we identify cyclic adenosine diphosphoribose (cADPR) as an agonist of TRPM2 with dual activity: at concentrations above 100 microM, cADPR can gate the channel by itself, whereas lower concentrations of 10 microM have a potentiating effect that enables ADPR to gate the channel at nanomolar concentrations. ADPR's breakdown product adenosine monophosphate (AMP) specifically inhibits ADPR, but not cADPR-mediated gating of TRPM2, whereas the cADPR antagonist 8-Br-cADPR exhibits the reverse block specificity. Our results establish TRPM2 as a coincidence detector for ADPR and cADPR signaling and provide a functional context for cADPR as a second messenger for Ca2+ influx.

Respiring mitochondria maintain a membrane potential (⌬⌿) 1 of Ϫ150 to Ϫ180 mV (⌬⌿, inside negative). This high ⌬⌿ constitutes a large driving force for the electrophoretic influx of cations, either through specific channels or by diffusion through the membrane. Several cation channels have been characterized physiologically (reviewed in Refs. 1 and 2), and recently, a single one has been identified molecularly (3). These transport systems seem to have intrinsic control mechanisms which ensure that the matrix cation concentrations stay within physiological ranges, far below chemical equilibrium.Diffusive permeability of the inner mitochondrial membrane to ions is generally low but physiologically significant, as it lowers the pH gradient and membrane potential. Moreover, if not counteracted by extrusion, steadily increasing concentrations of matrix cations (and of compensating anions) will lead to an imbalance of osmotic pressure across the inner mitochondrial membrane. As a consequence, water will pass through the membrane, causing excessive swelling and eventual rupture of the organelle (1, 2, 4).As first proposed by P. Mitchell (5), mitochondria have carrier systems allowing the electroneutral exchange of cations against H ϩ (and anions against OH Ϫ ). These exchangers counteract the ⌬⌿-driven cation leakage of the membrane and also cation imbalances due to changes in mitochondrial physiology. Mitochondrial cation distribution is, therefore, a steady state, in which the accumulation ratio is modulated by the relative rates of cation influx and efflux by means of separate pathways.Many physiological studies have been devoted to cation/H ϩ exchange systems (reviewed in Ref.

Steady-state concentrations of mitochondrial Mg 2+ previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains. While its structural and functional similarity to the bacterial Mg 2+ transport protein CorA suggested a role for Mrs2p in Mg 2+ in¯ux into the organelle, other functions in cation homeostasis could not be excluded. Making use of the¯uorescent dye mag-fura 2 to measure free Mg 2+ concentrations continuously, we describe here a high capacity, rapid Mg 2+ in¯ux system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Dy and inhibited by cobalt(III)hexaammine. Overexpression of Mrs2p increases in¯ux rates 5-fold, while the deletion of the MRS2 gene abolishes this high capacity Mg 2+ in¯ux. Mg 2+ ef¯ux from isolated mitochondria, observed with low Dy only, also requires the presence of Mrs2p. Cross-linking experiments revealed the presence of Mrs2p-containing complexes in the mitochondrial membrane, probably constituting Mrs2p homooligomers. Taken together, these ®ndings characterize Mrs2p as the ®rst molecularly identi®ed metal ion channel protein in the inner mitochondrial membrane.

TRPM2 (previously designated TRPC7 or LTRPC2) is a Ca2+-permeable nonselective cation channel that contains a C-terminal enzymatic domain with pyrophosphatase activity, which specifically binds ADP-ribose. Cyclic ADP-ribose (cADPR) and hydrogen peroxide (H2O2) can facilitate ADPR-mediated activation of heterologously expressed TRPM2. Here, we show that the two Ca2+-mobilizing second messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) strongly activate natively expressed TRPM2 channels in Jurkat T cells. TRPM2 activation by both agonists can be partially suppressed by the ADPR antagonist adenosine monophosphate (AMP), which suggests that cADPR and NAADP lead to mobilization of endogenous ADPR presumably via metabolic conversion. The remaining channel activity is due to direct gating of TRPM2 by the two agonists and can be completely suppressed by 8-Br-cADPR, which suggests that cADPR and NAADP share a common binding site on TRPM2 that can regulate TRPM2 activity in synergy with ADPR. We conclude that cADPR and NAADP, in combination with ADPR, represent physiological co-activators of TRPM2 that contribute to Ca2+ influx in T lymphocytes and presumably other cell types that express this channel.

The molecular biology of mammalian magnesium transporters and their interrelations in cellular magnesium homeostasis are largely unknown. Recently, the mouse SLC41A1 protein was suggested to be a candidate magnesium transporter with channel-like properties when overexpressed in Xenopus laevis oocytes. Here, we demonstrate that human SLC41A1 overexpressed in HEK293 cells forms protein complexes and locates to the plasma membrane without, however, giving rise to any detectable magnesium currents during whole cell patch clamp experiments. Nevertheless, in a strain of Salmonella enterica exhibiting disruption of all three distinct magnesium transport systems (CorA, MgtA, and MgtB), overexpression of human SLC41A1 functionally substitutes these transporters and restores the growth of the mutant bacteria at magnesium concentrations otherwise non-permissive for growth. Thus, we have identified human SLC41A1 as being a bona fide magnesium transporter. Most importantly, overexpressed SLC41A1 provide HEK293 cells with an increased magnesium efflux capacity. With outwardly directed Mg(2+) gradients, a SLC41A1-dependent reduction of the free intracellular magnesium concentration accompanied by a significant net decrease of the total cellular magnesium concentration could be observed in such cells. SLC41A1 activity is temperature-sensitive but not sensitive to the only known magnesium channel blocker, cobalt(III) hexaammine. Taken together, these data functionally identify SLC41A1 as a mammalian carrier mediating magnesium efflux.

Magnesium (Mg(2+)), the second most abundant divalent intracellular cation, is involved in the vast majority of intracellular processes, including the synthesis of nucleic acids, proteins, and energy metabolism. The concentration of intracellular free Mg(2+) ([Mg(2+)](i)) in mammalian cells is therefore tightly regulated to its optimum, mainly by an exchange of intracellular Mg(2+) for extracellular Na(+). Despite the importance of this process for cellular Mg(2+) homeostasis, the gene(s) encoding for the functional Na(+)/Mg(2+) exchanger is (are) still unknown. Here, using the fluorescent probe mag-fura 2 to measure [Mg(2+)](i) changes, we examine Mg(2+) extrusion from hSLC41A1-overexpressing human embryonic kidney (HEK)-293 cells. A three- to fourfold elevation of [Mg(2+)](i) was accompanied by a five- to ninefold increase of Mg(2+) efflux. The latter was strictly dependent on extracellular Na(+) and reduced by 91% after complete replacement of Na(+) with N-methyl-d-glucamine. Imipramine and quinidine, known unspecific Na(+)/Mg(2+) exchanger inhibitors, led to a strong 88% to 100% inhibition of hSLC41A1-related Mg(2+) extrusion. In addition, our data show regulation of the transport activity via phosphorylation by cAMP-dependent protein kinase A. As these are the typical characteristics of a Na(+)/Mg(2+) exchanger, we conclude that the human SLC41A1 gene encodes for the Na(+)/Mg(2+) exchanger, the predominant Mg(2+) efflux system. Based on this finding, the analysis of Na(+)/Mg(2+) exchanger regulation and its involvement in the pathogenesis of diseases such as Parkinson's disease and hypertension at the molecular level should now be possible.

The product of the nuclear MRS2 gene, Mrs2p, is the only candidate splicing factor essential for all group II introns in mitochondria of the yeast Saccharomyces cerevisiae. It has been shown to be an integral protein of the inner mitochondrial membrane, structurally and functionally related to the bacterial CorA Mg(2+) transporter. Here we show that mutant alleles of the MRS2 gene as well as overexpression of this gene both increase intramitochondrial Mg(2+) concentrations and compensate for splicing defects of group II introns in mit(-) mutants M1301 and B-loop. Yet, covariation of Mg(2+) concentrations and splicing is similarly seen when some other genes affecting mitochondrial Mg(2+) concentrations are overexpressed in an mrs2Delta mutant, indicating that not the Mrs2 protein per se but certain Mg(2+) concentrations are essential for group II intron splicing. This critical role of Mg(2+) concentrations for splicing is further documented by our observation that pre-mRNAs, accumulated in mitochondria isolated from mutants, efficiently undergo splicing in organello when these mitochondria are incubated in the presence of 10 mM external Mg(2+) (mit(-) M1301) and an ionophore (mrs2Delta). This finding of an exceptional sensitivity of group II intron splicing toward Mg(2+) concentrations in vivo is unprecedented and raises the question of the role of Mg(2+) in other RNA-catalyzed reactions in vivo. It explains finally why protein factors modulating Mg(2+) homeostasis had been identified in genetic screens for bona fide RNA splicing factors.

Parkinson's disease (PD) is a complex multifactorial ailment predetermined by the interplay of various environmental and genetic factors. Systemic and intracellular magnesium (Mg) deficiency has long been suspected to contribute to the development and progress of PD and other neurodegenerative diseases. However, the molecular background is unknown. Interestingly, gene SLC41A1 located in the novel PD locus PARK16 has recently been identified as being a Na+/Mg2+ exchanger (NME, Mg2+ efflux system), a key component of cellular magnesium homeostasis. Here, we demonstrate that the substitution p.A350V potentially associated with PD is a gain-of-function mutation that enhances a core function of SLC41A1, namely Na+-dependent Mg2+ efflux by 69±10% under our experimental conditions (10-minute incubation in high-Na+ (145 mM) and completely Mg2+-free medium). The increased efflux capacity is accompanied by an insensitivity of mutant NME to cAMP stimulation suggesting disturbed hormonal regulation and leads to a reduced proliferation rate in p.A350V compared with wt cells. We hypothesize that enhanced Mg2+-efflux conducted by SLC41A1 variant p.A350V might result, in the long-term, in chronic intracellular Mg2+-deficiency, a condition that is found in various brain regions of PD patients and that exacerbates processes triggering neuronal damage.