Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii.

Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [gamma-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [gamma-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ of Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol in 2 mM. Both ADP and cAMP inhibit the reaction.     

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system.

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

The hydrolysis of triphosphoinositide by a phosphodiesterase in rat kidney cortex

The hydrolysis of triphosphoinositide by a phosphodiesterase has been demonstrated in rat kidney cortex. Subcellular fractionation studies revealed that the enzyme activity was predominantly found in the supernatant fraction. After acid precipitation and ammonium sulfate fractionation, the soluble enzyme was free from triphosphoinositide phosphomonoesterase activity.Although the partially purified enzyme did not require added divalent cations for activity, it was strongly inhibited by EDTA (0.1 mm). In the absence of EDTA, added MgCl₂ or CaCl₂ depressed the enzyme activity. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol and other phospholipids. It hydrolyzed both diphosphoinositide and triphosphoinositide with the formation of 1,2-diglyceride and organic phosphate.     

Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes

1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.     

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[gamma-32P]ATP. The Km's for 32P incorporation into di- and triphosphoinositide were 110 and 48 microM ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 degrees C, whereas the optimum for triphosphoinositide labeling occurred at 43 degrees C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 degrees C but not at 25 degrees C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 mM MgCl2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl2 (5 to 50 mM) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (less than or equal to 100 mM) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 mM NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 mM MgCl2 and 200 mM NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 mM MgCl2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.     

Studies on the properties of triphosphoinositide phosphomonoesterase and phosphodiesterase of rabbit iris smooth muscle.

The rabbit iris smooth muscle has been shown to contain triphosphoinositide phosphomonoesterase (phosphatidyl-myo-inositol-4,5-bisphosphate phosphohydrolase, EC 3.1.3.36) and phosphodiesterase (triphosphoinositide inositoltrisphosphohydrolase, EC 3.1.4.11) activities. Under our experimental conditions about 77% of the phosphomonoesterase and 61% of the phosphodiesterase activities were localized in the particulate fraction. The kinetic properties of the enzymes in the microsomal fraction were examined. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol under the present assay condition. The effects of Ca2+ and Mg2+ were also studied. Although the microsomal enzymes did not require added divalent cations for their activities, both the phosphomonoesterase and phosphodiesterase were appreciably inhibited by 1 mM EDTA. Phosphodiesterase and phosphomonoesterase were stimulated by Ca2+ and Mg2+, respectively. The demonstration of triphosphoinositide phosphodiesterase in the iris muscle, coupled with the findings that this enzyme is activated by Ca2+ and is not influenced by acetylcholine add further support to our previous conclusion (J. Pharmacol. Exp. Ther. (1978) 204, 655--668; J. Neurochem. (1978) 30, 517--525) that an increased Ca2+ influx, following the interaction between the neurotransmitter and its receptor, could act to stimulate the phosphodiesterase, thus leading to increased triphosphoinositide breakdown and increased phosphatidic acid via increased diacylglycerol.     

THE BIOSYNTHESIS OF TRIPHOSPHOINOSITIDE BY PURIFIED MYELIN OF PERIPHERAL NERVE

The distribution of diphosphoinositide kinase activity in homogenates and myelin of rabbit nerve was determined by measuring the synthesis of labelled triphosphoinositide. Evidence is presented to show that the biosynthesis of triphosphoinositide in peripheral myelin involves a membrane-bound diphosphoinositide kinase.     

ENZYMES OF PHOSPHOINOSITIDE METABOLISM DURING RAT BRAIN DEVELOPMENT

—The activities of four enzymes concerned with inositol lipid metabolism have been determined in homogenates of rat brains of different ages. The enzymes are CDP-diglyceride inositol phosphatidate transferase, phosphatidylinositol kinase, diphosphoinositide kinase and triphosphoinositide phosphomonoesterase. The activities of all the enzymes increased with age. Phosphatidylinositol kinase activity rose most sharply well before myelination, reaching a maximum at about 6 days of age. Diphosphoinositide kinase and triphosphoinositide phosphomonoesterase activities increased most rapidly during myelination. The increase in CDP-diglyceride inositol phosphatidate transferase showed no definite association with any period of development. It is concluded that triphosphoinositide metabolism is associated with myelin or a closely related structure.     

Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization.

The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.     

Demonstration of protein kinase activities in the coronary artery of cattle]

Protein kinase activities were identified in a soluble and a particulate fraction from the A. coronaria of cattle. For both protein kinase activities Mg++ is essential. Protamine was used as a substrate of the protein kinase activity of the soluble fraction. The pH optimum of the protein kinase activity of the soluble fraction is around 6.5. The Km-value of the protein kinase for ATP is 1.9 +/- 0.4 - 10(-5) M. cAMP stimulates the protein kinase activity more effectively than cGMP. Ca++ cannot replace Mg++; monovalent cations (Na+ and K+) show no influence. The protein kinase activity of the fraction was determined via endogenous phosphorylation. By means of the cAMP-dependent particulate protein kinase 72 to 80 percent of the serine residues are phosphorylated. The pH optimum of the protein kinase activity of the particulate fraction lies around 7.0. The Km-value of the enzyme for ATP is 6.6 +/- 0.8 - 10(-5) M. cGMP stimulates the protein kinase of the particulate fraction better than cAMP. For the protein kinase activity of this fraction Ca++ replaces Mg++ in the endogenous phosphorylation but not in the exogenous phosphorylation (protamine). In the presence of Mg++ and in the additional presence of Na+ or K+, the protein kinase activity is suppressed in the endogenous phosphorylation whereas it is stimulated in the exogenous phosphorylation.     

The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg(2+)-dependent and is stimulated by Mn(2+) and F(-) ions. The initial reaction is rapid, with a broad pH profile and a ;transition' temperature for its activation energy at 27 degrees C. The apparent K(m) for ATP is 5mum. ATP, N-ethylmaleimide, Cu(2+) ions and NaIO(4) are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2-3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.     

Polyphosphoinositides in myelin

1. On fractionation of guinea-pig forebrain homogenates by differential and gradient-density centrifugation most of the polyphosphoinositides were recovered in the myelin-rich particles. 2. The phospholipids of pure preparations of myelin contained di- and tri-phosphoinositide in proportions 2-3 times greater than in the whole-brain phospholipids. 3. Di- and tri-phosphoinositide appeared in young rat brain during the period of myelination. 4. After the administration of [(32)P]phosphate to guinea pigs the labelling of the polyphosphoinositides in isolated pure myelin was as great as in the whole brain, whereas little synthesis of the other myelin phospholipids had occurred. 5. When brain subcellular fractions were incubated with [gamma-(32)P]ATP, some triphosphoinositide labelling occurred in the myelin-rich fraction whereas the active labelling of diphosphoinositide was localized mainly in the mitochondrial fraction. 6. The Na(+), K(+) and Mg(2+) plus Ca(2+) concentrations in purified myelin have been determined. The Mg(2+) plus Ca(2+) content present showed close acid-base equivalence to the polyphosphoinositides. 7. It is concluded that di- and tri-phosphoinositide are rapidly-metabolizing components of the myelin sheath or intimately associated structures.     

Polyphosphoinositide synthesis in rabbit erythrocyte membranes

Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm [γ-³²P]ATP and MgCl₂ results in incorporation of ³²P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol ³²P/mg/h, respectively. Incorporation of ³²P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with ³²P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of ³²P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl₂ and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.     

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non-ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity. Developmental changes were the same for both kinase activities, which were low in unmyelinated brain and sciatic nerve. The activities correlated with the concentration of polyphosphoinositides in chick brain where they increased 4-5 fold during the period of active myelination and remained high in the mature brain. The kinase activities correlated with the rate of triphosphoinositide deposition in sciatic nerve. Following a 2-3 fold increase during the initial phase of myelination the activities declined to values as low as those of embryonic nerve.     

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate.

1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction.     

The phosphatidylinositol kinase of rat brain

1. The presence of a phosphatidylinositol kinase in homogenates of adult rat brain was shown by using labelled ATP or labelled phosphatidylinositol. 2. The kinase was activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+), Cu(2+), K(+), Na(+) and F(-). 3. The detergents sodium deoxycholate, Cutscum and Triton X-100 markedly stimulated the reaction; sodium taurocholate, Tween-20 and cetyltrimethyl-ammonium bromide were less effective. 4. The activity of the enzyme was dependent on SH groups. 5. The subcellular distribution of the kinase in brain resembled that of Na(+)-plus-K(+)-stimulated adenosine triphosphatase and 5'-nucleotidase.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

Characterization of the protein kinase activity of TRPM7/ChaK1, a protein kinase fused to the transient receptor potential ion channel

Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called alpha-kinases that display no sequence homology to conventional protein kinases. It is an unusual bifunctional protein that contains an alpha-kinase domain fused to an ion channel. The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis. However, little is known about its protein kinase activity. To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria. ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues. The kinase is specific for ATP and cannot use GTP as a substrate. ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin. Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity. The kinase requires Mg(2+) (optimum at 4-10 mM) or Mn(2+) (optimum at 3-5 mM), with activity in the presence of Mn(2+) being 2 orders of magnitude higher than in the presence of Mg(2+). Zn(2+) and Co(2+) inhibited ChaK1-cat kinase activity. Ca(2+) at concentrations up to 1 mM did not affect kinase activity. Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg(2+) can directly modulate TRPM7/ChaK1 kinase activity in vivo.