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• W2023164919 abstract "Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-β (Aβ) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1. We show that CALHM1 encodes a multipass transmembrane glycoprotein that controls cytosolic Ca2+ concentrations and Aβ levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca2+ conductance across the plasma membrane. Importantly, we determined that the CALHM1 P86L polymorphism (rs2986017) is significantly associated with AD in independent case-control studies of 3404 participants (allele-specific OR = 1.44, p = 2 × 10−10). We further found that the P86L polymorphism increases Aβ levels by interfering with CALHM1-mediated Ca2+ permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca2+ channel that controls Aβ levels and susceptibility to late-onset AD. Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-β (Aβ) peptide deposition. Using TissueInfo to screen for genes preferentially expressed in the hippocampus and located in AD linkage regions, we identified a gene on 10q24.33 that we call CALHM1. We show that CALHM1 encodes a multipass transmembrane glycoprotein that controls cytosolic Ca2+ concentrations and Aβ levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca2+ conductance across the plasma membrane. Importantly, we determined that the CALHM1 P86L polymorphism (rs2986017) is significantly associated with AD in independent case-control studies of 3404 participants (allele-specific OR = 1.44, p = 2 × 10−10). We further found that the P86L polymorphism increases Aβ levels by interfering with CALHM1-mediated Ca2+ permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca2+ channel that controls Aβ levels and susceptibility to late-onset AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a massive loss of neurons in several brain regions and by the presence of cerebral senile plaques comprised of aggregated amyloid-β (Aβ) peptides (Mattson, 2004Mattson M.P. Pathways towards and away from Alzheimer's disease.Nature. 2004; 430: 631-639Crossref PubMed Scopus (2306) Google Scholar, Selkoe, 2001Selkoe D.J. Alzheimer's disease: Genes, proteins, and therapy.Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (4909) Google Scholar). The first atrophy observed in the AD brain occurs in the medial temporal lobe, which includes the hippocampus, and is the result of a massive synaptic degeneration and neuronal death (Braak and Braak, 1991Braak H. Braak E. Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol. (Berl.). 1991; 82: 239-259Crossref PubMed Scopus (10466) Google Scholar, de Leon et al., 2007de Leon M.J. Mosconi L. Blennow K. DeSanti S. Zinkowski R. Mehta P.D. Pratico D. Tsui W. Saint Louis L.A. Sobanska L. et al.Imaging and CSF studies in the preclinical diagnosis of Alzheimer's disease.Ann. N Y Acad. Sci. 2007; 1097: 114-145Crossref PubMed Scopus (170) Google Scholar). Two major Aβ species are found, Aβ40 and Aβ42; both are produced from the sequential endoproteolysis of the amyloid precursor protein (APP) by BACE1/β-secretase and by presenilin (PS)/γ-secretase complexes. APP can also undergo a nonamyloidogenic proteolysis by α-secretase, which cleaves APP within the Aβ sequence and thereby precludes Aβ generation (Marambaud and Robakis, 2005Marambaud P. Robakis N.K. Genetic and molecular aspects of Alzheimer's disease shed light on new mechanisms of transcriptional regulation.Genes Brain Behav. 2005; 4: 134-146Crossref PubMed Scopus (59) Google Scholar, Wilquet and De Strooper, 2004Wilquet V. De Strooper B. Amyloid-beta precursor protein processing in neurodegeneration.Curr. Opin. Neurobiol. 2004; 14: 582-588Crossref PubMed Scopus (191) Google Scholar). The etiology of the disease is complex because of its strong genetic heterogeneity. Rare autosomal-dominant mutations in the genes encoding APP, PS1, and PS2 cause early-onset AD, whereas complex interactions among different genetic variants and environmental factors are believed to modulate the risk for the vast majority of late-onset AD (LOAD) cases (Kennedy et al., 2003Kennedy J.L. Farrer L.A. Andreasen N.C. Mayeux R. St George-Hyslop P. The genetics of adult-onset neuropsychiatric disease: Complexities and conundra?.Science. 2003; 302: 822-826Crossref PubMed Scopus (140) Google Scholar, Lambert and Amouyel, 2007Lambert J.C. Amouyel P. Genetic heterogeneity of Alzheimer's disease: Complexity and advances.Psychoneuroendocrinology. 2007; 32: S62-S70Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Pastor and Goate, 2004Pastor P. Goate A.M. Molecular genetics of Alzheimer's disease.Curr. Psychiatry Rep. 2004; 6: 125-133Crossref PubMed Scopus (57) Google Scholar). To date, the only susceptibility gene unambiguously demonstrated worldwide is the ɛ4 allele of APOE on chromosome 19 (Strittmatter et al., 1993Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Apolipoprotein E: High-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1993; 90: 1977-1981Crossref PubMed Scopus (3520) Google Scholar). However, epidemiological studies indicate that the presence of the APOE ɛ4 allele cannot explain the overall heritability of AD, implying that a significant proportion of LOAD cases is attributable to additional genetic risk factors (Lambert and Amouyel, 2007Lambert J.C. Amouyel P. Genetic heterogeneity of Alzheimer's disease: Complexity and advances.Psychoneuroendocrinology. 2007; 32: S62-S70Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Pastor and Goate, 2004Pastor P. Goate A.M. Molecular genetics of Alzheimer's disease.Curr. Psychiatry Rep. 2004; 6: 125-133Crossref PubMed Scopus (57) Google Scholar). Supporting this observation, concordant evidence of linkage to LOAD has been observed in different chromosomal regions, including on chromosome 10, where a strong and consensual susceptibility locus is present (Bertram et al., 2000Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. et al.Evidence for genetic linkage of Alzheimer's disease to chromosome 10q.Science. 2000; 290: 2302-2303Crossref PubMed Scopus (447) Google Scholar, Blacker et al., 2003Blacker D. Bertram L. Saunders A.J. Moscarillo T.J. Albert M.S. Wiener H. Perry R.T. Collins J.S. Harrell L.E. Go R.C. et al.Results of a high-resolution genome screen of 437 Alzheimer's disease families.Hum. Mol. Genet. 2003; 12: 23-32Crossref PubMed Scopus (300) Google Scholar, Ertekin-Taner et al., 2000Ertekin-Taner N. Graff-Radford N. Younkin L.H. Eckman C. Baker M. Adamson J. Ronald J. Blangero J. Hutton M. Younkin S.G. Linkage of plasma Abeta42 to a quantitative locus on chromosome 10 in late-onset Alzheimer's disease pedigrees.Science. 2000; 290: 2303-2304Crossref PubMed Scopus (319) Google Scholar, Farrer et al., 2003Farrer L.A. Bowirrat A. Friedland R.P. Waraska K. Korczyn A.D. Baldwin C.T. Identification of multiple loci for Alzheimer disease in a consanguineous Israeli-Arab community.Hum. Mol. Genet. 2003; 12: 415-422Crossref PubMed Scopus (116) Google Scholar, Kehoe et al., 1999Kehoe P. Wavrant-De Vrieze F. Crook R. Wu W.S. Holmans P. Fenton I. Spurlock G. Norton N. Williams H. Williams N. et al.A full genome scan for late onset Alzheimer's disease.Hum. Mol. Genet. 1999; 8: 237-245Crossref PubMed Scopus (321) Google Scholar, Myers et al., 2000Myers A. Holmans P. Marshall H. Kwon J. Meyer D. Ramic D. Shears S. Booth J. DeVrieze F.W. Crook R. et al.Susceptibility locus for Alzheimer's disease on chromosome 10.Science. 2000; 290: 2304-2305Crossref PubMed Scopus (340) Google Scholar). However, despite intensive research efforts to characterize the genetic factor(s) located within the chromosome 10 region, no gene has been conclusively linked to LOAD risk (Bertram et al., 2006Bertram L. Hsiao M. Lange C. Blacker D. Tanzi R.E. Single-nucleotide polymorphism rs498055 on chromosome 10q24 is not associated with Alzheimer disease in two independent family samples.Am. J. Hum. Genet. 2006; 79: 180-183Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, Grupe et al., 2006Grupe A. Li Y. Rowland C. Nowotny P. Hinrichs A.L. Smemo S. Kauwe J.S. Maxwell T.J. Cherny S. Doil L. et al.A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease.Am. J. Hum. Genet. 2006; 78: 78-88Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Kuwano et al., 2006Kuwano R. Miyashita A. Arai H. Asada T. Imagawa M. Shoji M. Higuchi S. Urakami K. Kakita A. Takahashi H. et al.Dynamin-binding protein gene on chromosome 10q is associated with late-onset Alzheimer's disease.Hum. Mol. Genet. 2006; 15: 2170-2182Crossref PubMed Scopus (59) Google Scholar, Minster et al., 2006Minster R.L. DeKosky S.T. Kamboh M.I. Lack of association of two chromosome 10q24 SNPs with Alzheimer's disease.Neurosci. Lett. 2006; 408: 170-172Crossref PubMed Scopus (5) Google Scholar). A number of neurodegenerative disorders are caused by mutations in genes expressed principally in the central nervous system. This is the case for the brain proteins tau and α-synuclein, which are linked to autosomal-dominant forms of frontotemporal dementia and Parkinson's disease, respectively. Here, we postulated that susceptibility to LOAD could come from genes predominantly expressed in affected brain regions, such as the hippocampus. We used TissueInfo (Skrabanek and Campagne, 2001Skrabanek L. Campagne F. TissueInfo: High-throughput identification of tissue expression profiles and specificity.Nucleic Acids Res. 2001; 29 (E102–E102)Crossref PubMed Scopus (44) Google Scholar) and the Alzgene database (Bertram et al., 2007Bertram L. McQueen M.B. Mullin K. Blacker D. Tanzi R.E. Systematic meta-analyses of Alzheimer disease genetic association studies: The AlzGene database.Nat. Genet. 2007; 39: 17-23Crossref PubMed Scopus (1356) Google Scholar) to screen for genes predominantly expressed in the hippocampus and located in linkage regions for LOAD, and identified CALHM1, a gene of unknown function, located on chromosome 10 at 1.6 Mb of the LOAD marker D10S1671 (Bertram et al., 2000Bertram L. Blacker D. Mullin K. Keeney D. Jones J. Basu S. Yhu S. McInnis M.G. Go R.C. Vekrellis K. et al.Evidence for genetic linkage of Alzheimer's disease to chromosome 10q.Science. 2000; 290: 2302-2303Crossref PubMed Scopus (447) Google Scholar). We found that CALHM1 homomultimerizes, controls cytosolic Ca2+ concentrations, and shares similarities with the predicted selectivity filter of the N-methyl-D-aspartate receptor (NMDAR). Voltage-clamp analyses further revealed that CALHM1 generates Ca2+-selective cation currents at the plasma membrane. Importantly, we determined that the frequency of the rare allele of the nonsynonymous single-nucleotide polymorphism (SNP) rs2986017 in CALHM1, which results in a proline-to-leucine substitution at codon 86 (P86L), is significantly increased in AD cases in five independent cohorts. Further investigation demonstrated the functional significance of the rs2986017 SNP by showing that the P86L mutation promotes Aβ accumulation via a loss of CALHM1 control on Ca2+ permeability and cytosolic Ca2+ levels. Here, we propose that CALHM1 is a component of a previously uncharacterized cerebral Ca2+ channel family involved in Aβ metabolism and that CALHM1 variants may influence the risk for LOAD. We screened the human genome with TissueInfo to annotate human transcripts with tissue expression levels derived from the expressed sequence tag database (dbEST) (Campagne and Skrabanek, 2006Campagne F. Skrabanek L. Mining expressed sequence tags identifies cancer markers of clinical interest.BMC Bioinformatics. 2006; 7: 481Crossref PubMed Scopus (22) Google Scholar, Skrabanek and Campagne, 2001Skrabanek L. Campagne F. TissueInfo: High-throughput identification of tissue expression profiles and specificity.Nucleic Acids Res. 2001; 29 (E102–E102)Crossref PubMed Scopus (44) Google Scholar). Out of 33,249 human transcripts, the TissueInfo screen identified 30 transcripts, corresponding to 12 genes, with expression restricted to the hippocampus (Table 1). These transcripts matched either one or two ESTs sequenced from the hippocampus. Among these genes, one of unknown function, previously annotated as FAM26C, matched two hippocampal ESTs and mapped to the AD locus on 10q24.33 (Table 1). This gene, hereafter referred to as calcium homeostasis modulator 1 (CALHM1), encodes an open reading frame (ORF) of 346 amino acids and is predicted to contain four hydrophobic domains (HDs; TMHMM prediction) and two N-glycosylation motifs (NetNGlyc 1.0 prediction) (Figure 1A). No significant amino acid sequence homology to other functionally characterized proteins was found. Sequence database searches identified five human homologs of CALHM1 (collectively identified as the FAM26 gene family). Two homologs of human CALHM1 with broader tissue expression profiles (see the Supplemental Data available online) are clustered next to CALHM1 in 10q24.33 and are designated CALHM2 (26% protein sequence identity, previously annotated as FAM26B) and CALHM3 (39% identity, FAM26A) (Figure 1A). CALHM1 is conserved across at least 20 species, including mouse and C. elegans (Figures 1A and 1B).Table 1TissueInfo Expression ScreenChromosomeBandEnsembl Transcript IDHit(s)Hit(s) in HippocampusaIndicates how many ESTs matching the transcript were sequenced from a cDNA library made from the hippocampus.Tissue SummaryGene Name or Other ID1p34.3ENST0000031963722hippocampusEPHA102p21ENST0000030607821hippocampusKCNG32q37.1ENST0000031306421hippocampusC2orf526q15ENST0000030372631hippocampusCNR16q25.3ENST0000030825411hippocampusRetired in Ensembl 466q27ENST0000032258311hippocampusNP_787118.29q21.33ENST0000029874331hippocampusGAS110q24.33ENST0000032990532hippocampusCALHM1(FAM26C)11q24.1ENST0000035459731hippocampusOR8B317q25.3ENST0000032693121hippocampusQ8N8L1_HUMAN19p12ENST0000036088511hippocampusRetired in Ensembl 46Xq27.2ENST0000029829611hippocampusMAGEC3One transcript is shown for each gene identified in the screen. Genomic location and number of hit(s) in dbEST are reported for each transcript.a Indicates how many ESTs matching the transcript were sequenced from a cDNA library made from the hippocampus. Open table in a new tab One transcript is shown for each gene identified in the screen. Genomic location and number of hit(s) in dbEST are reported for each transcript. Using RT-PCR, we analyzed human CALHM1 expression in 20 tissues and six brain regions. The expression of CALHM1 was highest in the total adult brain and in all brain regions tested. CALHM1 expression was noticeably lower in all other tissues, including fetal brain (Figure 2A). qRT-PCR revealed endogenous CALHM1 expression in retinoic acid-differentiated SH-SY5Y cells (Figure S1A), suggesting that CALHM1 is a protein of neuronal origin. Immunofluorescence staining in transiently transfected cells revealed that CALHM1 strongly localized to the endoplasmic reticulum (ER), where it colocalized with the ER resident protein GRP78 (Figure 2B, left and middle panels). However, some cells revealed immunoreactivity for CALHM1 at the cell surface, suggesting that a pool of CALHM1 was localized at or near the plasma membrane (Figure 2B, right panel, arrows). Western blotting (WB) analyses revealed the presence of two immunoreactive bands in CALHM1-transfected cells (Figure 2C, lanes 1 and 2). Because human CALHM1 is predicted to be N-glycosylated at asparagine residues N74 and N140 (see Figure 1A, asterisks), we asked whether these bands might represent different N-glycosylated forms of the protein. Treatment of CALHM1-transfected cell lysates with N-glycosidase F, which cleaves all types of asparagine-bound N-glycans, completely eliminated the appearance of the higher molecular weight band and resulted in the accumulation of the lower band, which we conclude corresponds to the unmodified core protein (Figure 2C, lanes 2 and 4). CALHM1 was partially resistant to endoglycosidase H-mediated deglycosylation (Figure 2C, lanes 2 and 3), indicating that CALHM1 can reach the medial Golgi compartment and the cell surface, where proteins are terminally glycosylated and acquire resistance to endoglycosidase H. Plasma membrane expression of CALHM1 was also investigated by cell surface biotinylation. Figure 2D illustrates that a pool of CALHM1, enriched in glycosylated forms of the protein, was biotinylated and thus was present at the plasma membrane. We further determined that substitution of the N140 residue to alanine (N140A) completely prevented CALHM1 glycosylation, whereas N74A substitution had no effect (Figure 2C, lanes 5–7). Thus, CALHM1 is a multipass transmembrane protein, N-glycosylated at the residue N140, predominantly expressed in the adult brain, and localized to the ER and plasma membranes. These data further indicate that the HD3-HD4 loop, which contains the N140 residue, is oriented toward the luminal side when CALHM1 is in the ER membrane and toward the extracellular space when CALHM1 reaches the plasma membrane. The predicted membrane topology of CALHM1 suggests the presence of one re-entrant hydrophobic loop that does not cross the membrane bilayer and three membrane-spanning segments (TMHMM prediction). In the absence of significant homology to other characterized proteins, we postulated from the predicted topology that CALHM1 could function as an ion channel component. This is in part based on a suggestive similarity with the topology of ionotropic glutamate receptors, which also contain three transmembrane segments and a re-entrant loop that forms the lining of the ion channel pore region (Wollmuth and Sobolevsky, 2004Wollmuth L.P. Sobolevsky A.I. Structure and gating of the glutamate receptor ion channel.Trends Neurosci. 2004; 27: 321-328Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Because some ionotropic glutamate receptors are Ca2+-permeable membrane proteins (Gouaux and Mackinnon, 2005Gouaux E. Mackinnon R. Principles of selective ion transport in channels and pumps.Science. 2005; 310: 1461-1465Crossref PubMed Scopus (592) Google Scholar), we asked whether CALHM1 could control cytoplasmic Ca2+ levels. Measurements of intracellular Ca2+ concentration ([Ca2+]i) were conducted under resting conditions in the presence of physiological concentrations of extracellular Ca2+. To reveal possible changes in the rate of Ca2+ entry in CALHM1-expressing cells, [Ca2+]i measurements were also performed under extracellular “Ca2+ add-back” conditions. These conditions are obtained after a transient external Ca2+ depletion that generates a driving force for Ca2+ entry. When the Ca2+ fluorescent dye Fluo-4 was used in mouse hippocampal HT-22 cells, no robust changes in fluorescence measurements were found under resting conditions after CALHM1 expression (data not shown). However, CALHM1 expression resulted in a strong and sustained increase in [Ca2+]i after extracellular Ca2+ add back (Figure 3A). CALHM1 expression significantly increased the initial rate of change in [Ca2+]i producing a peak of fluorescence at ∼2 min after Ca2+ addition (Figures 3A and 3B, Peak). CALHM1 expression also induced a significant elevation in the steady-state [Ca2+]i, compared to control conditions (Figures 3A and 3B, Steady-state). To measure absolute [Ca2+]i, we also determined the effect of CALHM1 on [Ca2+]i by using the ratiometric Ca2+ indicator Fura-2. We confirmed that, under Ca2+ add-back conditions, CALHM1 expression induced a significant elevation of [Ca2+]i from 106 ± 4 nM (prior to Ca2+ add back) to 264 ± 48 nM at the peak (after Ca2+ addition), whereas control cells showed no significant changes in [Ca2+]i (from 105 ± 5 nM to 110 ± 6 nM; Figure S2). Because massive Ca2+ influx can be cytotoxic, we evaluated the viability of cells expressing CALHM1. Figure S3 illustrates that, in both normal and Ca2+ add-back conditions, no noticeable cell viability impairments or cytotoxicity were observed after CALHM1 expression. One important mechanism of Ca2+ entry coupled to ER Ca2+ release is called store-operated Ca2+ entry (SOCE). In excitable cells, such as neurons, voltage-gated Ca2+ channels (VGCCs) represent another critical mechanism of Ca2+ influx during membrane depolarization (Berridge et al., 2003Berridge M.J. Bootman M.D. Roderick H.L. Calcium signalling: Dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (3823) Google Scholar). Inhibition of SOCE by the use of 2-APB did not prevent CALHM1 from affecting [Ca2+]i (Figure 3C). Similarly, selective blockage of the different subtypes of VGCCs with SNX-482 (R type VGCC inhibitor), mibefradil (T type), nifedipine (L type), or ω-conotoxin MVIIC (N, P, Q types) did not block the rise of [Ca2+]i induced by CALHM1 expression (Figures 3D and 3E). Because cytosolic Ca2+ can be released from intracellular stores via activation of the inositol 1,4,5-triphosphate receptors (InsP3Rs) or the ryanodine receptors (RyRs) at the ER membrane (Berridge et al., 2003Berridge M.J. Bootman M.D. Roderick H.L. Calcium signalling: Dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (3823) Google Scholar), we next asked whether CALHM1 expression promotes InsP3R or RyR activation. The InsP3R inhibitor xestospongin C and the RyR inhibitor dantrolene were found to have no effect on the CALHM1-driven [Ca2+]i increase (Figure 3F), indicating that ER Ca2+ release via InsP3Rs or RyRs did not account for the effect of CALHM1 on cytosolic Ca2+ levels. Because presenilins were recently proposed to form ER calcium leak channels (Tu et al., 2006Tu H. Nelson O. Bezprozvanny A. Wang Z. Lee S.F. Hao Y.H. Serneels L. De Strooper B. Yu G. Bezprozvanny I. Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations.Cell. 2006; 126: 981-993Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), we also investigated whether CALHM1 requires the presence of PS1 or PS2 to control cytosolic Ca2+ levels. We found that CALHM1 expression caused similar increases in [Ca2+]i in WT fibroblasts and in fibroblasts deficient for both PS1 and PS2 (Figure S4), showing that CALHM1 controls cytosolic Ca2+ levels independently of presenilins. We found, however, that the increase of [Ca2+]i observed after CALHM1 expression was blocked by cobalt (Co2+) and nickel (Ni2+), two nonspecific Ca2+ channel blockers. Indeed, Figures 3G and 3H show that 50 μM Co2+ or 10 μM Ni2+ were sufficient to completely inhibit the rise of intracellular Ca2+ induced by CALHM1 without causing changes in CALHM1 expression (Figure 3I). Because Ni2+ does not penetrate the cells (Shibuya and Douglas, 1992Shibuya I. Douglas W.W. Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: Evidence provided by fluorescence changes in fura-2-loaded cells.Endocrinology. 1992; 131: 1936-1941Crossref PubMed Scopus (96) Google Scholar), these results suggest that the two inorganic Ca2+ channel blockers acted at the plasma membrane to block Ca2+ entry. Collectively, these results strongly indicate that CALHM1 expression promotes Ca2+ influx via activation of a cell-surface ion channel that is distinct from known VGCC or SOCE channels. Because many channels multimerize to form an ion pore, and because monomeric CALHM1 cannot create a functional pore with three transmembrane segments, we asked whether CALHM1 could form multimers. WB analyses of CALHM1-transfected cells under nonreducing conditions revealed the presence of immunoreactive bands with molecular weights compatible with dimers and tetramers of CALHM1 (Figure 4A). To test the possibility that CALHM1 self-associates, we coexpressed in cells two different tagged versions of the protein and used coimmunoprecipitation experiments to determine whether the two versions of CALHM1 form a complex. We found that immunoprecipitation of Myc-tagged CALHM1 coprecipitated V5-tagged CALHM1 (Figure 4B), indicating that CALHM1 homomultimerized to form dimeric and possibly tetrameric structures. Ionotropic glutamate receptors are ion-conducting membrane proteins with specific ion-selectivity properties (Gouaux and Mackinnon, 2005Gouaux E. Mackinnon R. Principles of selective ion transport in channels and pumps.Science. 2005; 310: 1461-1465Crossref PubMed Scopus (592) Google Scholar). Recent advances in the structural analysis of ion channels have determined that the ion selectivity of some ion channels is controlled by a short amino acid sequence called the selectivity filter, which forms a narrow constriction in the pore across the membrane bilayer (Gouaux and Mackinnon, 2005Gouaux E. Mackinnon R. Principles of selective ion transport in channels and pumps.Science. 2005; 310: 1461-1465Crossref PubMed Scopus (592) Google Scholar). The predicted selectivity filter of ionotropic glutamate receptors is located in a re-entrant loop called M2 and is critical for Ca2+ permeability (Dingledine et al., 1999Dingledine R. Borges K. Bowie D. Traynelis S.F. The glutamate receptor ion channels.Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, Wollmuth and Sobolevsky, 2004Wollmuth L.P. Sobolevsky A.I. Structure and gating of the glutamate receptor ion channel.Trends Neurosci. 2004; 27: 321-328Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). By manual inspection, we screened ionotropic glutamate receptor subunit sequences for similarities with CALHM1 and found a short sequence in C terminus of CALHM1 HD2 that aligns with the predicted ion-selectivity filter of NMDAR NR2 subunits (Figure 4C). Previous studies have determined that the asparagine (N) residue in the so-called Q/R/N site of NMDAR NR2 subunits is critical for ion selectivity and permeation (see Figure 4C, asterisk) (Wollmuth and Sobolevsky, 2004Wollmuth L.P. Sobolevsky A.I. Structure and gating of the glutamate receptor ion channel.Trends Neurosci. 2004; 27: 321-328Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). By sequence comparison, we identified a highly conserved N72 residue in human CALHM1 that aligns with the Q/R/N site at the C terminus end of the second hydrophobic domain of both CALHM1 and NMDAR (Figure 4C, asterisk). Importantly, we found that mutagenesis of the N72 residue to glycine (N72G) resulted in a significant inhibition of the effect of CALHM1 on [Ca2+]i (Figures 4D and 4E). Two-electrode voltage clamping of Xenopus oocytes was employed so that the effects of CALHM1 expression on plasma membrane conductance could be determined. Oocytes were injected with either water or CALHM1 cRNA, and conductance was recorded 24–72 hr later in a normal Na+-containing bath. In water-injected oocytes, the resting membrane potential Vm was −38mV ± 1mV (n = 74), and the membrane conductance, measured as the slope conductance around the reversal potential Vrev, was 1.5 ± 0.2 μS (n = 7). In contrast, the Vm in CALHM1-expressing oocytes was depolarized to −16mV ± 0.3mV (n = 96; p < 0. 0001), and membrane conductance was enhanced to 422 ± 78 μS (n = 12) (p < 0.005). The current-voltage (I-V) relation was outwardly rectifying (slope conductances of 372 ± 110 and 670 ± 131 μS at −55mV and +55mV, respectively) (Figure 4F). Depolarization of the resting Vm suggested that the CALHM1-enhanced conductance was contributed by a Na+ permeability. Isosmotic replacement of bath Na+ with NMDG hyperpolarized Vm by 7mV ± 0.8mV (Figure 4F; n = 12; p < 0.0001). These results demonstrate that expression of CALHM1 conferred a constitutive Na+ conductance in Xenopus oocyte plasma membrane. Expression of CALHM1 in CHO cells also generated an outwardly rectifying current in whole-cell recordings with Cs+ in the pipette (cytoplasmic) and Na+ in the bath (Figure 4G). The current reversed ∼0mV, indicating that the relative permeabilities of Cs+ and Na+ were similar (PNa: PCs = 0.8). The current was not observed in either untransfected or EGFP-transfected cells (Figure 4G, control), and it was eliminated when the monovalent cations in the bath and pipette solutions were replaced with NMDG (Figure 4H), indicating that the current was carried by Cs+ and Na+. The CALHM1-induced slope conductance measured around the Vrev was 360 ± 60 pS/pF (n = 42), compared with 74 ± 17 pS/pF (n = 11) in control cells. Gd3+ (100 μM) nearly completely inhibited the CALHM1-induced current (Figure 4G). With bath Na+ replaced by NMDG and 20 mM Ca2+, an outwardly rectifying, Gd3+-sensitive current was observed in the CALHM1-expressing cells that reversed at +8.3mV ± 2.9mV (n = 7), indicating PCa: PCs = 5 (Figure 4H). Thus, expression of CALHM1 conferred a constitutive Ca2+-selective cation current in CHO cell plasma membrane. In summary, our studies show that a region of CALHM1 shares sequence similarities with the selectivity filter of NMDAR and that the N72 residue is a key determinant in the control of cytosolic Ca2+ levels by CALHM1. Furthermore, electrophysiological analyses in CALHM1-expressing Xenopus oocytes and CHO cells demonstrated that CALHM1 i" @default.
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• W2023164919 title "A Polymorphism in CALHM1 Influences Ca2+ Homeostasis, Aβ Levels, and Alzheimer's Disease Risk" @default.
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