A TRPM7 Variant Shows Altered Sensitivity to Magnesium That May Contribute to the Pathogenesis of Two Guamanian Neurodegenerative Disorders

Home , TRPM7

A TRPM7 variant shows altered sensitivity to magnesium that may contribute to the pathogenesis of two Guamanian neurodegenerative disorders

Meredith C. Hermosura*†, Hannah Nayakanti*, Maxim V. Dorovkov‡, Fernanda R. Calderon*, Alexey G. Ryazanov‡, David S. Haymer§, and Ralph M. Garruto¶

*Bekesy Laboratory of Neurobiology, Paciﬁc Biosciences Research Center, and §Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822; ‡Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and ¶Laboratory of Biomedical Anthropology and Neurosciences, Binghamton University, State University of New York, Binghamton, NY 13902-6000

Contributed by Ralph M. Garruto, June 19, 2005 Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism shown to be significantly permeable to transition metal ions in dementia (PD-G) have been epidemiologically linked to an envi- addition to Ca2ϩ and Mg2ϩ and was suggested to be involved in ,ronment severely deﬁcient in calcium (Ca2؉) and magnesium the homeostatic regulation of these ions (11–14). In addition Mg2؉). Transient receptor potential melastatin 7 (TRPM7) is a TRPM7 has been implicated in anoxic neuronal death by) bifunctional protein containing both channel and kinase domains mediating IOGD, a cation current activated during prolonged that has been proposed to be involved in the homeostatic regu- oxygen–glucose deprivation (OGD) (15). That study also lation of intracellular Ca2؉,Mg2؉, and trace metal ion concentra- showed that low levels of Ca2ϩ and Mg2ϩ enhanced TRPM7͞ tion. There is evidence that TRPM7 is constitutively active and that IOGD currents in neurons. Given the established association the number of available channels is dependent on intracellular free between ALS-G and PD-G disease incidence with an environ- Mg2؉ levels. We found a TRPM7 variant in a subset of ALS-G and ment deficient in Ca2ϩ and Mg2ϩ, we wondered whether TRPM7 PD-G patients that produces a protein with a missense mutation, was somehow involved. For example, it is possible that genetic T1482I. Recombinant T1482I TRPM7 exhibits the same kinase cat- variants of TRPM7 produce channel proteins with slightly altered alytic activity as WT TRPM7. However, heterologously expressed function. The altered channel might function adequately under T1482I TRPM7 produces functional channels that show an in- normal conditions but might be seriously compromised in a creased sensitivity to inhibition by intracellular Mg2؉. Because the conducive situation such as an environment deficient in Ca2ϩ incidence of ALS-G and PD-G has been associated with prolonged and Mg2ϩ. exposure to an environment severely deﬁcient in Ca2؉ and Mg2؉, Here, we report the identification of a TRPM7 variant in a we propose that this variant TRPM7 allele confers a susceptibility subset of ALS-G and PD-G patients that causes a missense genotype in such an environment. This study represents an initial mutation, changing Thr-1482 to isoleucine (Ile). In functional attempt to address the important issue of gene–environment studies, we show that the channel protein encoded by the variant interactions in the etiology of these diseases. is more sensitive to inhibition by internal free Mg2ϩ.We interpret our results in terms of the Ca2ϩ- and Mg2ϩ-deficient amyotrophic lateral sclerosis ͉ calcium ͉ gene–environment environment that has been epidemiologically linked to these interactions ͉ phosphorylation ͉ parkinsonism dementia diseases. To our knowledge, T1482I is the first exon variant identified in connection with ALS-G and PD-G and the first uamanian amyotrophic lateral sclerosis (ALS-G) and par- evidence of a functional alteration in a physiologically relevant Gkinsonism dementia (PD-G) are distinct but related neu- ion channel in a population at high risk for the development of rodegenerative disorders found in high incidence on the Western ALS and parkinsonism dementia. Like many other complex Pacific Islands of Guam and Rota (1). Despite intensive inves- diseases, ALS-G and PD-G likely have a multifactorial etiology. tigation, a clear understanding of the etiology and pathogenesis The T1482I variant could be one of many contributory factors. of these disorders remains elusive. Most evidence now suggests Methods that a complex interplay between genetic susceptibility and exposure to certain environmental factors is involved (2–4). The Specimens, DNA Extraction, and Sequencing. Pathologically con- genetic susceptibility hypothesis is supported by observations firmed brain autopsy specimens include affected and age- that ALS-G and PD-G cases cluster in families and that siblings, matched controls who died of nonneurological problems. There parents, and offspring of afflicted patients are at increased risk are 9 PD-G (4 males, 5 females), 13 ALS-G (including 4 classical sporadic cases) (8 males, 5 females), and 23 control subjects (11 for developing these diseases (4, 5). Epidemiological and animal Ϫ studies have identified two candidate environmental triggers: males, 12 females). The samples have been stored at 80°C in toxins from a traditional food source, the cycad plant (6), and R.M.G.’s laboratory and were sent to the University of Hawaii altered mineral content of the soil and drinking water (1, 7). in a liquid N2 shipper. Genomic DNA was extracted from the Prolonged exposure to an environment low in Ca2ϩ and Mg2ϩ samples by using the DNeasy tissue DNA extraction kit (Qiagen, Valencia, CA). Primers were designed based on the published and high in bioavailable aluminum, manganese, or other toxic ࿝ metals was proposed to have led to the development of three human sequence for TRPM7 (NC 000015) by the PRIMER 3 input high-incidence foci of ALS-G and PD-G in the Western Pacific:

in the Mariana Islands, in the Kii Peninsula of Japan, and in Abbreviations: ALS-G, Guamanian amyotrophic lateral sclerosis; PD-G, Guamanian parkin- southern West New Guinea (1, 7–9). sonism dementia; TRP, transient receptor potential; TRPM7, TRP melastatin 7; hTRPM7, Transient receptor potential melastatin 7 (TRPM7) is a human TRPM7; DOX, doxycycline; HA, hemagglutinin. ubiquitously expressed member of the TRP superfamily of ion Data deposition: The SNP data reported in this paper have been deposited in the National channels. Because TRP channels are primarily classified based Center for Biotechnology Information SNP database (dbSNP ID no. rs8042919). on sequence homology, these channels exhibit a wide range of †To whom correspondence should be addressed. E-mail: [email protected]. biophysical properties and function (10). TRPM7 has been © 2005 by The National Academy of Sciences of the USA

11510–11515 ͉ PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505149102 Downloaded by guest on September 27, 2021 software (http:͞͞frodo.wi.mit.edu). Each primer pair typically added as needed. The pH was adjusted to 7.2, and osmolarity was encompasses intron–exon boundaries unless the exon is too long measured (normally 300–315 mOsm). Free Mg2ϩ concentrations (Ͼ400 bp). In this case, more than one pair is designed. We were were calculated by using MAXCHELATOR (16). careful to include enough overlap between primers to allow for Patch-clamp experiments were performed in the tight-seal, unreliable sequence readouts near priming areas. Primer se- whole-cell configuration at 23–26°C. Patch pipettes made of quences are available on request. PCR products were run on a borosilicate glass (Kimax, Kimble Glass, Vineland, NJ) had gel, excised, gene-cleaned (GeneClean Kit III, MP Biomedicals͞ resistances between 2 and 4 MU` when filled with standard Qbiogene, Irvine, CA), and sent to the University of Hawaii intracellular solution. Currents were filtered at 2.9 kHz and Biotech Core Facility for BigDye terminator cycle sequencing digitized at 100-␮s intervals with a computer-based amplifier (Applied Biosystems Prism377, Applied Biosystems). Samples system, EPC-9 (HEKA Electronics, Lambrecht͞Pfalz, Ger- were sequenced in both directions. Resulting chromatograms many). Voltages were corrected for a liquid junction potential of were viewed with the EDITVIEW software (version 1.0.1, Applied 10 mV between external and internal solutions. Capacitive Biosystems) and aligned against the published TRPM7 genomic currents and series resistance were corrected by using the and mRNA reference sequences (NC࿝000015 and NM࿝017672, automatic capacitance compensation of the EPC-9. Immediately respectively) using VECTOR NTI (Informax, Bethesda). after whole-cell break-in, 50-ms voltage ramps from Ϫ100 mV to ϩ100 mV were delivered from a holding potential of 0 mV every Constructs, Mutagenesis, and Creation of Stable Cell Line. The tet- 2 s for the duration of the experiment (typically 10 min). The racycline͞doxycycline (DOX)-inducible HEK-293 cell line sta- time course of current development was measured at Ϫ80 mV bly expressing WT human TRPM7 (hTRPM7) and the expres- and ϩ80 mV. Measurements were then exported to IGOR PRO sion construct hTRPM7͞pCDNA4͞TO were gifts from A. (version 5.03, WaveMetrics, Lake Oswego, OR) for further Scharenberg, A. Perraud, and C. Schmitz (14). This recombinant analysis. Where applicable, statistical errors of averaged data are hTRPM7 is tagged with the hemagglutinin (HA) epitope. We given as the mean Ϯ SEM with n determinations. created the T1482I mutation in the hTRPM7͞pCDNA4͞TO construct by using Strategene’s QuikChange site-directed mu- Expression, Purification, Activity Assays, and Phosphoamino Acid tagenesis kit. The entire construct was sequenced to verify the Analysis of Human WT TRPM7 Kinase and T1482I TRPM7 Kinase (T1482I presence of the desired mutation and absence of any unwanted Kinase). We expressed and purified WT TRPM7 kinase and mutations. To create an inducible cell line expressing T1482I T1482I kinase by using the methods described in ref. 17. Briefly, mutant channels, we transfected HEK-293 stably expressing the the C terminus of TRPM7 containing the last 462 amino acids tetracycline repressor (TR-293 cell line, Invitrogen) by using (1403–1864) was produced as a fusion with maltose-binding Lipofectamine 2000 (Invitrogen) and selected for stable trans- protein. In WT TRPM7 kinase, this fusion protein has Thr-1482; fectants by zeocin treatment (400 ␮g͞ml). Inducible TRPM7 in T1482I kinase, Thr-1482 was replaced with Ile. expression was tested by performing RT-PCR on RNA tran- Assays for kinase activity were carried out in a total reaction scripts extracted from cells that have been exposed to 1 ␮g͞ml volume of 25 ␮l of assay buffer containing 50 mM Hepes–KOH DOX for 24 h. RNA concentrations were adjusted so that the (pH 7.4), 10 mM MgCl2, 4 mM MnCl2, 100 ␮MATP,2␮Ci of same amount was used in the RT-PCR reactions. [␥-33P]ATP (specific activity of 3,000 Ci͞mmol; 1 Ci ϭ 37 GBq), and 0.5 ␮g of fusion protein. After incubation at 30°C for 10 min, Cell Culture. Cells stably expressing WT or T1482I TRPM7 were Laemmli sample buffer was added and the samples were boiled grown on glass coverslips at 37°C in DMEM plus 10% FBS, 5 and then analyzed by SDS͞PAGE and autoradiography. ␮g͞ml blasticidin, and 0.4 mg͞ml zeocin in the presence of 5% Phosphoamino acid analysis was performed by using methods CO2 and passaged twice a week. TRPM7 expression was induced described in ref. 17. Briefly, phosphoamino acids were separated by the addition of 1 ␮g͞ml DOX to the culture medium. All by 2D electrophoresis on thin-layer cellulose plates 10 ϫ 10 cm experiments were performed on cells that have been exposed to (cellulose on polyester, Aldrich). The first-dimension separation DOX for 24–48 h. was at 1,000 V for 20 min in an electrophoresis buffer containing 0.58 M formic acid and 1.36 M acetic acid (pH 1.9). The Immunofluorescent Staining. Induced cells were washed with PBS, second-dimension separation was at 1,000 V for 8 min in pH 3.5 fixed in 4% formaldehyde solution, and permeabilized with 0.1% electrophoresis buffer containing 0.87 M acetic acid, 0.5% Triton X-100. Nonspecific staining was blocked by preincubation (vol͞vol) pyridine, and 0.5 mM EDTA. The TLC plates were with 10% goat serum before the addition of the primary antibody stained with 0.2% ninhydrin in ethanol and then exposed to film. (mouse anti-HA, Santa Cruz Biotechnology). The primary A phosphoimager (PhosphorImager Cyclone, PerkinElmer) was antibody was omitted for control cells. The cells were washed and used to quantify the phosphorylation levels of threonine and then stained with FITC-conjugated goat anti-mouse IgG. After serine residues. NEUROSCIENCE a final wash, the coverslips were mounted on glass slides with Ultra Cruz mounting medium (Santa Cruz Biotechnology) and Results and Discussion viewed by using an inverted microscope (IX51, Olympus, In a blind study, we searched for mutations in the TRPM7 gene Melville, NY) equipped with a xenon lamp, FITC emission by using genomic DNA extracted from brain autopsy tissues that filters, and a ϫ40 oil-immersion objective. Images were acquired were collected as part of the National Institutes of Health by using OPENLAB software (version 4.01, Improvision, Lexing- (National Institute of Neurological Disorders and Stroke) case ton, MA) and a charge-coupled device camera (ORCA IEEE ascertainment program for ALS-G and PD-G. The pathologi- 1394, Hamamatsu, Middlesex, NJ). All reagents were diluted in cally confirmed samples were from both men and women of PBS with 1.5% goat serum. Chamorro heritage, the indigenous people of Guam, and consist of 13 ALS-G, 9 PD-G, and 23 age-matched controls (death not Electrophysiology. Cells were kept in a modified Ringer’s solution linked to either ALS-G or PD-G). We found a nonsynonymous of the following composition: 140 mM NaCl, 2.8 mM KCl, 10 variant that causes a missense mutation, changing Thr-1482 to mM Hepes–NaOH, 1.5 mM CaCl2, 2 mM MgCl2, 5 mM CsCl, Ile-1482 in two ALS-G and three PD-G patients (Fig. 1). This and 10 mM glucose; osmolarity, 295–315 mOsm. The standard variant was not detected in any of the control Chamorro internal (pipette) solution contained 140 mM cesium-glutamate, specimens. 8 mM NaCl, 10 mM Hepes–CsOH, 10 mM CsEGTA, and 2.5 TRPM7 is a dual-function protein, possessing an ion-channel 2ϩ mM CaCl2. Calculated amounts of 1 M stock Mg solution were domain fused to a serine͞threonine ␣-kinase domain (Fig. 2a).

Hermosura et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11511 Downloaded by guest on September 27, 2021 Fig. 1. Chromatograms of ALS-G, PD-G, and control sequences. A heterozygous C-to-T transition (arrowhead) was found in ALS-G and PD-G samples but not in controls. This transition causes an exon mutation where Thr is replaced with Ile (box). Sequences are in the reverse (minus-strand) orientation. Num- bers in parentheses are numeric sample codes used in the blind study.

␣-Kinases are characterized by their unique ability to phosphorylate substrates within ␣-helices (18, 19), as opposed to conven- tional protein kinases, which phosphorylate their substrates within loops, ␤-turns, and irregular structures (20). Thr-1482 lies between the channel and kinase domains. Computer analysis (JPRED, http͞͞www.compbio.dundee.ac.uk͞ϳwww-jpred) of the secondary structure of this region predicts Thr-1482 to be part of an ␣-helix and therefore a potential substrate for autophosphorylation (Fig. 2b). By contrast, Ile-1482 is predicted to form part of a coil. We have determined that Thr-1482 is evolutionarily conserved and is present in fish, amphibian, avian, Fig. 2. Thr-1482 is in an ␣-helix between the channel and kinase domains and canine, and primate species (Fig. 2c). In murine TRPM7, is evolutionarily conserved. (a) Schematic representation of TRPM7. Residue 1482 is found between the coiled-coil and kinase domains. (b) Secondary Thr-1482 is replaced by serine, which also is a potential substrate structure analysis predicts Thr-1482 as part of an ␣-helix, whereas Ile-1482 of TRPM7 kinase. The evolutionary conservation suggests that becomes part of a coil. (c) Alignment of the appropriate region of TRPM7 from phosphorylation of the residue in this position is important for different species shows evolutionary conservation of Thr-1482 (boxed), except normal function. Because Ile cannot be phosphorylated, one in the mouse, where serine (Ser) is present instead. National Center for could predict that there would be a noticeable functional con- Biotechnology Information (GenBank, Entrez Protein) accession numbers are sequence in TRPM7 channels produced by the variant allele. shown in the right. Orangutan and Xenopus sequences were translated from To investigate possible functional differences between WT ESTs CR769569 and CA973711. Sequences were aligned by using CLUSTALW (http:͞͞www.ch.embnet.org͞software͞clustalw.html). and T1482I channels, we created a DOX-inducible HEK-293 cell line stably expressing hTRPM7 with the mutated residue, Ile- 1482. Except for the presence of this mutation, this cell line is antibody, we compared the localization of expressed WT and similar to one stably expressing WT hTRPM7 (14), thereby T1482I channels. We observed the same pattern of punctate facilitating direct comparison. We verified inducible channel membrane and cytoplasmic staining in both WT- and T1482I- expression by RT-PCR and selected the clone where T1482I overexpressing cells, suggesting that the mutation does not affect expression level was similar to WT when grown in the presence channel trafficking (Fig. 3c). of the inducer, DOX, for 24 h (Fig. 3a). Faint bands representing Next, we compared WT and T1482I channel function by low-level expression of native TRPM7 can be detected in HEK- whole-cell patch clamp. Induced cells kept in a bath solution 293 in the absence of DOX. In T1482I-transfected cells, ex- containing near physiological levels of Ca2ϩ and Mg2ϩ were pressed channels could be chimeras made up of native and perfused with a pipette solution where no Mg2ϩ was added mutant components. To show that the recombinant channel is by (nominal 0 Mg2ϩ) to elicit maximal TRPM7 currents (14, 21). far the dominant species expressed in induced cells, we se- Under these conditions, WT- and T1482I-expressing cells quenced RT-PCR products. Only mutant transcripts were de- showed the characteristic TRPM7 current͞voltage (I͞V) rela- tected by the sequencing reaction in T1482I-transfected cells tionship upon break-in (t ϭ 0), which increases in size as exposed to DOX (Fig. 3b). intracellular Mg2ϩ is removed during the course of perfusion Before carrying out functional studies, we examined the (Fig. 4a). The presence of TRPM7-mediated currents at break-in cellular localization of expressed WT and T1482I channel pro- makes the important point that a small population of WT and teins. It could be possible that T1482I channels exhibit altered T1482I channels is open in resting cells. The time course of localization and trafficking patterns, and this, in turn, could current development in WT- and T1482I-expressing cells shows directly affect whole-cell currents measured in patch-clamp that steady-state is reached within 5 min in both cases (Fig. 4b, experiments. Using immunofluorescent staining with anti-HA filled triangles for 0 nominal Mg2ϩ). These results show that the

11512 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505149102 Hermosura et al. Downloaded by guest on September 27, 2021 Fig. 3. Assessment of inducible expression and immunolocalization of expressed WT and T1482I. (a) RT-PCR of inducible HEK-293 cells stably transfected with WT and T1482I in the presence and absence of the inducer, DOX. The mutant clone selected exhibits channel expression levels that closely match WT expression after induction. Faint bands detected in the absence of DOX represent low-level expression of endogenous TRPM7. (b) Sequence chromatograms of the RT-PCR products from induced cells in a confirm the genotype of the expressed channels (arrowheads). Primers for the plus strand were used for the sequencing reactions. (c) Anti-HA immunofluorescent staining of HEK-293 cells induced to express WT and T1482I channels. The same pattern of punctate membrane and cytoplasmic staining indicates that the mutation does not alter channel trafficking and localization.

T1482I channel is functional, mediating currents with the same pronounced outward rectification as WT. There are, however, Fig. 4. Whole-cell currents and sensitivity to internal Mg2ϩ.(a) Representa- some noticeable differences in the currents elicited by the tive whole-cell currents elicited by a 50-ms voltage ramp from Ϫ100 mV to ϩ nominal 0 Mg2ϩ solution in cells expressing WT and T1482I 100 mV in cells overexpressing WT and T1482I perfused with nominal 0 Mg2ϩ ϭ channels. Peak current size is larger and activation time is slightly pipette solution. Data traces taken at whole-cell break-in (t 0), 5 min, and 10 min into the experiment are shown. All recordings were measured in cells faster for WT. The mean (ϮSEM) peak current density in cells Ϯ ͞ ͞ that have been exposed to DOX for 24–36 h. Small currents with the pro- expressing WT is 179 43 pA pF (picoamp picofarad), com- nounced outward rectiﬁcation characteristic for TRPM7 can be detected at pared with 102 Ϯ 18 pA͞pF for their mutant counterparts. The break-in. The currents developed as cells were dialyzed, reaching near- time course for half-maximal activation (t1/2max) is Ϸ42 s for maximal levels within 3 min. These results show that T1482I channels are WT, compared with 62 s for T1482I. functional. (b) Time course of development of WT and T1482I currents in the 2ϩ Collectively, these results suggest that T1482I channels are presence of various concentrations of internal Mg . Plots represent average current densities measured at ϩ 80 mV for each Mg2ϩ concentration used: WT, either less readily activated or more sensitive to inhibition. It is 0mMMg2ϩ, n ϭ 8; 0.5 mM Mg2ϩ, n ϭ 6;1mMMg2ϩ, n ϭ 7;2mMMg2ϩ, n ϭ NEUROSCIENCE known that TRPM7 is sensitive to suppression by intracellular 10;4mMMg2ϩ, n ϭ 6;and6mMMg2ϩ, n ϭ 6; T1482I, 0 mM Mg2ϩ, n ϭ 15; 0.5 ϩ free Mg2 (14, 21, 22). The mechanism underlying this inhibition mM Mg2ϩ, n ϭ 5;1mMMg2ϩ, n ϭ 12;2mMMg2ϩ, n ϭ 5;4mMMg2ϩ, n ϭ 5; remains unknown, but the kinase domain is thought to be and6mMMg2ϩ, n ϭ 4. Averaged traces were normalized to the break-in value involved. Residue 1482 is located Ͻ100 residues upstream of the at0Mg2ϩ.(c) T1482I channels are more susceptible to inhibition by internal Mg2ϩ. Current densities measured at Ϫ80 mV and ϩ80 mV are plotted as a kinase domain, close enough for the T1482I mutation to influ- 2ϩ 2ϩ function of pipette Mg concentration. The data points were obtained by ence how the channel responds to Mg . To address this issue, subtracting the break-in value measured in each cell from the maximum 2ϩ we compared Mg sensitivity by measuring whole-cell currents current density attained during the ﬁrst 400 s of the experiment and averaging in WT- and T1482I-expressing cells dialyzed with pipette solu- the results for each Mg2ϩ concentration used (ϮSEM). tions containing different concentrations of added Mg2ϩ.By ϩ using MAXCHELATOR (15), the free Mg2 concentrations in these solutions were calculated as follows: 0.5 mM added Mg2ϩ (free and 1.0 mM in many cell types (23), although free Mg2ϩ in Mg2ϩ ϭ 367 ␮M); 1 mM added Mg2ϩ (free Mg2ϩ ϭ 737 ␮M); neurons was measured to range between 400 and 800 ␮M (24). 2 mM added Mg2ϩ (free Mg2ϩ ϭ 1.48 mM); 4 mM added Mg2ϩ For comparison purposes, averaged currents plotted in Fig. 4b (free Mg2ϩ ϭ 3.03 mM); and 6 mM added Mg2ϩ (free Mg2ϩ ϭ are normalized to the break-in value at 0 Mg2ϩ. Our results show 4.61 mM). For reference, the physiological concentration of T1482I to be more sensitive to Mg2ϩ inhibition when this ion is intracellular free Mg2ϩ has been estimated to vary between 0.5 present at concentrations Ͻ2 mM, which includes the physio-

Hermosura et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11513 Downloaded by guest on September 27, 2021 TRPM7 channels. More studies are definitely needed to clarify the mechanism involved. As shown here and in earlier studies (12–14, 22), TRPM7 is constitutively active albeit regulated by intracellular free Mg2ϩ. Therefore, the number of available channels at any given time is influenced by existing intracellular Mg2ϩ levels. T1482I is more sensitive to Mg2ϩ inhibition when this ion is present in the critical physiological range, making it likely that fewer T1482I channels are available in normal resting conditions. Because TRPM7 is thought to be involved in maintaining homeostatic levels of Ca2ϩ,Mg2ϩ, and trace metal ions, cells expressing the mutant channel will have a higher likelihood of becoming deficient in these ions than cells expressing WT. Prolonged exposure to an environment deficient in Ca2ϩ and Mg2ϩ such as that found in Fig. 5. Comparison of kinase catalytic activity of WT and T1482I mutant the Western Pacific could have unfavorable consequences. In TRPM7 and phosphoamino acid analysis. (a) Puriﬁed recombinant human this case, cells heterozygous for the variant could eventually TRPM7 (amino acids 1403–1864) and T1482I mutant were incubated with become severely deficient in Ca2ϩ and Mg2ϩ and become [␥-33P]ATP in the kinase reaction mixture as described in Methods. The samples ͞ vulnerable to the deleterious effects of such deficiencies. were analyzed by SDS PAGE and autoradiography. (b) Phosphoamino acid Ca2ϩ and Mg2ϩ deficiencies have been associated with neu- analysis of autophosphorylated WT TRPM7 and T1482I mutant (amino acids 1403–1864). Phosphoamino acid analysis was performed by hydrolysis of ropathology. In a study aimed at investigating effects of chronic phosphoproteins with HCl, separation of amino acids by using 2D electro- dietary calcium deficiency, monkeys maintained on a controlled phoresis on TLC plates, and autoradiography (see Methods). Quantitation of low-calcium diet showed neuropathological changes that were the amount of radioactivity incorporated into phosphoserine and phospho- compatible with those seen in early ALS-G and PD-G, such as threonine was performed by using a PhosphorImager. accumulation of phosphorylated neurofilaments, neurofibrillary tangles, and mitochondrial degeneration (25). Guamanian Chamorros afflicted with ALS-G and PD-G were found to have 2ϩ logical range for Mg . In the presence of 0.5 and 1.0 mM added hypocalcemia and reduced cortical bone mass (26). Brain and 2ϩ ␮ ␮ 2ϩ Mg (367 M and 737 M free Mg , respectively), WT spinal cord of ALS patients from the Kii Peninsula had signif- Ϸ currents are 20% less than the maximum obtainable (in icantly lower magnesium levels than did neurologically normal 2ϩ nominal 0 Mg ). T1482I currents, however, are more severely controls (26). It is possible that prolonged exposure to low affected. Under the same conditions, the peak current is only calcium could upset the delicate balance between Ca2ϩ- 2ϩ about half the size of that measured in nominal 0 Mg . These dependent ATP production and the controlled uptake and results are also documented in Fig. 4C where the relationship 2ϩ ϩ export of Ca from the mitochondria. The resulting imbalance between Mg2 dose and peak current density at ϩ80 mV and in intracellular Ca2ϩ levels could affect a myriad of Ca2ϩ- Ϸ80 mV is plotted. In WT, a graded reduction of peak currents dependent processes, including those affecting cytoskeletal or- occurred when 0.5 mM and 1 mM Mg2ϩ is added to the nominal ϩ ganization, generation of reactive oxygen species, and cell death. 0Mg2 solution. By contrast, peak T1482I current was sharply 2ϩ ϩ Mg is an essential cofactor of many metabolic enzymes and is reduced on addition of 0.5 mM Mg2 . No difference is seen in 2ϩ ϩ needed to bind cellular ATP (23). Mg deficiency, therefore, the presence of 2 mM Mg2 and higher. could also be detrimental, especially in highly metabolically It is important to understand how the replacement of Thr- active cells such as neurons. ϩ 1482 with Ile changes sensitivity to Mg2 . In an earlier study, Two of us have recently identified annexin 1 as a substrate for mutations that disrupt kinase activity also changed TRPM7’s TRPM7 kinase (28). Annexin 1 belongs to the annexin family of ϩ sensitivity to Mg2 inhibition (14). We wondered if the increased Ca2ϩ-regulated phospholipid binding proteins that are part of ϩ Mg2 sensitivity of T1482I channels is associated with changes the Ca2ϩ-signaling network in cells and is implicated in the in kinase activity. Using standard autophosphorylation kinase regulation of cell growth, cell death, and cellular response to assays (17), we compared the activities of WT TRPM7 kinase oxidative stress (reviewed in ref. 29). Because TRPM7 also has and T1482I kinase (Fig. 5a). We found that the catalytic activity been implicated in these processes, it is possible that TRPM7 and of T1482I is similar to that of WT, which indicates that the annexin 1 could act synergistically. Annexin 1 is Ca2ϩ-dependent ϩ increased sensitivity of T1482I channels to Mg2 is mediated and could be particularly vulnerable to the effects of Ca2ϩ through a different mechanism. deficiency. In light of the evolutionary conservation of Thr-1482, we The link between an environment severely deficient in Ca2ϩ examined whether it is autophosphorylated by comparing phos- and Mg2ϩ and the incidence of ALS-G and PD-G has been well phoamino acid patterns of WT and T1482I (Fig. 5b). Incorpo- established epidemiologically, but the molecular mechanism that ration of 33P into phosphothreonine was considerably less in could explain such an association has not been sufficiently T1482I, compared with WT. Quantitation of the amount of investigated so far. This study is an initial attempt in this radioactivity incorporated into phosphoserine and phospho- direction. We propose that the T1482I TRPM7 variant confers a threonine by using a PhosphorImager revealed that in WT, susceptibility genotype where the deleterious effect is unmasked Ϸ70% of radioactive phosphate is incorporated into phospho- after prolonged exposure to an environment severely deficient in serine and Ϸ30% is incorporated into phosphothreonine. How- Ca2ϩ and Mg2ϩ such as found in the Western Pacific (7–9). In ever, in T1482I, Ϸ80% of radioactive phosphate is incorporated southern Guam, where the incidence of ALS-G and PD-G have into phosphoserine, and 20% is incorporated into phosphothreo- been highest, Ca2ϩ levels in the soil and traditional spring nine. These results suggest that TRPM7 can indeed phosphor- drinking water were 10- to 100-fold lower than in other regions ylate itself at Thr-1482 and that this phosphorylation contributes of the island. Mg2ϩ in the water supply is similarly low, about Ϸone-third of all autophosphorylation at threonine residues. 10-fold lower in the South. This hypothesis also helps explain the Thr-1482 is located in a serine͞threonine-rich region of un- observed decline of ALS-G and PD-G in recent years in parallel known function. The increase in Mg2ϩ sensitivity of T1482I with modernization (6, 30) with the accompanying changes in channels seems to suggest that phosphorylation of Thr-1482 diet as well as the establishment of a treated water supply system. somehow affects the process whereby internal Mg2ϩ inhibits A check of the SNP database reveals that the T1482I variant of

11514 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505149102 Hermosura et al. Downloaded by guest on September 27, 2021 the TRPM7 gene was identified in other populations (Reference The question of how severe Ca2ϩ and Mg2ϩ deficiency leads SNP ID no. rs8042919) studied by Perlegen (http:͞͞www.ncbi. to neuronal damage remains to be more thoroughly explored. nih.gov͞SNP) and the International HapMap Project (http:͞͞ Although controlled low-calcium diets caused neuropathological www.hapmap.org). Samples from four populations were in- changes in monkeys (25), it is highly likely that ALS-G and PD-G cluded in the HapMap project: CEU (Utah residents with result from a multitude of factors, especially because we did not ancestry from northern and western Europe), YRI (Yoruba in find T1482I in all of the afflicted samples that were analyzed in Ibandan, Nigeria); HCB (Han Chinese in Beijing), and JPT this study. Potential contributory factors include other suscep- (Japanese in Tokyo). The genotype frequencies for rs8042919 tibility genes (31), mitochondrial mutations (32), and environ- ͞ ϭ ͞ ϭ ͞ ϭ mental toxins such as transition metals (33) and the cycad toxin are as follows: CEU (G G 0.850, A G 0.133, A A 0.017); ␤ ͞ ϭ ͞ ϭ ͞ ϭ ͞ ϭ -N-methylamino-L-alanine (34), perhaps interacting in a back- YRI (G G 1.000); HCB (G G 0.867, A G 0.111, A A ground of severe Ca2ϩ and Mg2ϩ deficiency. 0.022); and JPT (G͞G ϭ 0.591, A͞G ϭ 0.364, A͞A ϭ 0.045). No information regarding the health status of individuals in these We thank Drs. I. Cooke, K. Lum, M. Rayner, S. Thompson, Z. Urban, sample sources was available. However, it is interesting to note and R. Yanagihara for helpful advice and assistance; Dr. M. Alam, T. that the frequencies from Japan are much different from those Freitas, and S. Togashi for the secondary structure predictions; Dr. C. ϩ from the other three populations with an A͞G frequency of Patton for free Mg2 calculations; Drs. A. Scharenberg, A. Perraud, and ͞ 0.364 and an A͞A frequency of 0.045. Similarly, it is interesting C. Schmitz for the WT HA-hTRPM7 cell line and HA-WT hTRPM7 pCDNA4͞TO cDNA; and Ms. Laura Soloway for assistance with case to note that, like Guam, Japan has a well known high-incidence selection and tissue preparation. This work was supported by National focus of ALS and PD in the Kii Peninsula, located in an Institutes of Health Grants S11 NS043462 (to M.C.H.), and R01 environment low in Ca2ϩ and Mg2ϩ (9). GM057300 (to A.G.R.).

1. Garruto, R. M. (1991) Neurotoxicology 9, 347–378. 18. Ryazanov, A. G., Pavor, K. S. & Dorovkov, M. V. (1999) Curr. Biol. 9, R43–R45. 2. Bailey-Wilson, J. E., Plato, C. C., Elston, R. C. & Garruto, R. M. (1992) Am. J. 19. Drennan, D. & Ryazanov, A. G. (2004) Prog. Biophys. Mol. Biol. 85, 1–3. Med. Genet. 45, 68–76. 20. Pinna, L. A. & Ruzzene, M. (1996) Biochim. Biophys. Acta 1314, 191–225. 3. Plato, C. C., Garruto, R. M., Galasko, D., Craig, U. K, Plato, M., Gamst, A., 21. Hermosura, M. C., Monteilh-Zoller, M. K., Scharenberg, A. M., Penner, R. & Torres, J. M. & Wiederholt, W. (2003) Am. J. Epidemiol. 157, 149–157. Fleig, A. (2002) J. Physiol. 539, 445–458. 4. Plato, C. C., Reed, D. M., Elizan, T. S. & Kurland, L. T. (1967) Am. J. Hum. 22. Kerschbaum, H. H, Kozak, J. A. & Cahalan, M. D. (2003) Biophys. J. 84, Genet. 19, 617–632. 2293–2305. 5. Plato, C. C., Galasko, D., Garruto, R. M., Plato, M., Gamst, A., Torres, J. M. 23. Romani, A. & Scarpa, A. (1992) Arch. Biochem. Biophys. 298, 1–12. & Wiederholt, W. (2002) Neurology 58, 765–773. 24. Altura, B. M., Gebrewold, A., Zhang, A. & Altura, B. T. (2003) Neurosci. Lett. 6. Spencer, P. S., Nunn, P. B., Hugon, J., Ludolph, A. C., Ross, S. M., Roy, D. N. 341, 189–192. & Robertson, R. C. (1987) Science 237, 517–522. 25. Garruto, R. M., Shankar, S. K., Yanagihara, R., Salazar, A. M., Amyx, H. L. 7. Garruto, R. M., Yanagihara, R., Gajdusek, D. C. & Arion, D. M. (1984) in & Gajdusek, D. C. (1989) Acta Neuropathol. 78, 210–219. Amyotrophic Lateral Sclerosis in Asia and Oceania, eds. Chen, K. M. & Yase, 26. Yanagihara, R., Garruto, R. M., Gajdusek, D. C., Tomita, A., Uchikawa, T., Y. (National Taiwan Univ., Taiwan), pp. 265–329. Konagaya, Y., Chen, K. M., Sobue, I., Plato, C. C. & Gibbs, C. J., Jr. (1984) Ann. 8. Gajdusek, D. C. & Salazar, A. M. (1982) Neurology 32, 107–126. Neurol. 15, 42–48. 9. Garruto, R. M. & Yase, Y. (1986) Trends Neurosci. 9, 368–374. 27. Yasui, M., Ota, K. & Yoshida, M. (1997) Magn. Reson. Med. 10, 39–50. 10. Clapham, D. E. (2003) Nature 426, 817–824. 28. Dorovkov, M. V. & Ryazanov, A. G. (2004) J. Biol. Chem. 279, 50643–50646. 11. Runnels, L. W., Yue, L. & Clapham, D. E. (2001) Science 291, 1043–1047. 29. Gerke, V. & Moss, S. E. (2001) Physiol. Rev. 82, 331–371. 12. Nadler, M. J., Hermosura, M. C., Inabe, K., Perraud, A. L., Zhu, Q., Stokes, A. J., Kurosaki, T., Kinet, J. P., Penner, R., Scharenberg, A. M. & Fleig, A. 30. Garruto, R. M., Yanagihara, R. & Gajdusek, D. C. (1985) Neurology 35, (2001) Nature 411, 590–595. 193–198. 13. Monteilh-Zoller, M. K., Hermosura, M. C., Nadler, M. J., Scharenberg, A. M., 31. Poorkaj, P., Tsuang, D., Wijsman, E., Steinbart, E., Garruto, R.M., Craig, Penner, R. & Fleig, A. (2003) J. Gen. Physiol. 121, 49–60. U.-K., Chapman, N. H., Anderson, L., Bird, T. D., Plato, C. C., et al. (2001) 14. Schmitz, C., Perraud, A. L., Johnson, C. O., Inabe, K., Smith, M. K., Penner, Arch. Neurol. 58, 1871–1878. R., Kurosaki, T., Fleig, A. & Scharenberg, A. M. (2003) Cell 114, 191–200. 32. Coskun, P. E., Beal, M. F. & Wallace, D. C. (2004) Proc. Natl. Acad. Sci. USA 15. Aarts, M., Iihara, K., Wei, W. L., Xiong, Z. G., Arundine, M., Cerwinski, W., 101, 10726–10731. MacDonald, J. F. & Tymianski, M. (2003) Cell 115, 863–877. 33. Gellein, K., Garruto, R. M., Syversen, T., Sjobakk, T. E. & Flaten, T. P. (2003) 16. Patton, C., Thompson, S. & Epel, D. (2004) Cell Calcium 35, 427–431. Biol. Trace Elem. Res. 96, 39–60. 17. Ryazanova, L. V., Dorovkov, M. V., Ansari, A. & Ryazanov, A. G. (2004) 34. Cox, P. A., Banack, S. A. & Murch, S. J. (2003) Proc. Natl. Acad. Sci. USA 100, J. Biol. Chem. 279, 3708–3716. 13380–13383. NEUROSCIENCE

Hermosura et al. PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11515 Downloaded by guest on September 27, 2021