International audienceTRPM2 is a Ca 2+}-permeable cation channel gated by ADP-ribose (ADPR) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC protocol to determine ADPR contents in perchloric acid cell extracts. The reversed phase ion-pair HPLC protocol with a Mg 2+} containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were about 5 {mu}M in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP. We furthermore analyzed intracellular concentrations of cyclic ADPR (cADPR) with a cyclase assay involving enzymatic conversion of cADPR to NAD and fluorometric determination of NAD. Intracellular cADPR concentrations were about 0.2 {mu}M and not altered by fMLP. In patch-clamp experiments, ADPR (0.1 to 100 {mu}M) was dialyzed into granulocytes to analyze its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (< 10 nM) or a high (1 {mu}M) intracellular Ca 2+} concentration. TRPM2 currents were significantly larger in high than in low Ca 2+} (e.g. -225 ± 27.1 vs. -7 ± 2.0 pA/pF at 5 {mu}M ADPR) but no currents at all were observed in the absence of ADPR (ADPR concentration <= 0.3 {mu}M). cADPR (0.1, 0.3 and 10 {mu}M) was without effect even in the presence of subthreshold ADPR (0.1 {mu}M). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca 2+}. Thus, ADPR and Ca 2+} in concert behave as a messenger system for agonist-induced influx of Ca 2+} through TRPM2 in granulocytes

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.

TRPM2 channels play an important role in the activation process of neutrophil granulocytes. One mechanism of TRPM2 channel gating is the binding of intracellular ADP ribose (ADPR) to the Nudix box domain in the C-terminal tail of TRPM2. Intracellular Ca 2+ , although not an activator of TRPM2 by its own, significantly enhances TRPM2 gating by ADPR. Stimulation of neutrophil granulocytes with the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) induces release of Ca 2+ ions from intracellular stores which in cooperation with endogenous ADPR levels enable Ca 2+

No abstract