Cryo-EM structure of the heptameric calcium homeostasis modulator 1 channel

Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca²⁺-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the “down” position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the “down” position to the “up” position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel.     

Extracellular Chloride Modulates the Desensitization Kinetics of Acid-sensing Ion Channel 1a (ASIC1a)

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl⁻ in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl⁻ binding is unknown. We investigated the effect of Cl⁻ substitution on heterologously expressed ASIC1a current and H⁺-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl⁻ with the impermeable and inert anion methanesulfonate (MeSO₃⁻) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br⁻, I⁻, and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl⁻-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl⁻.     

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na⁺ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na⁺ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ_E455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β_E446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na⁺ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.     

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I _CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I _CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I _CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I _CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.     

Effect of the CALHM1 G330D and R154H Human Variants on the Control of Cytosolic Ca2+ and Aβ Levels

CALHM1 is a plasma membrane voltage-gated Ca²⁺-permeable ion channel that controls amyloid-β (Aβ) metabolism and is potentially involved in the onset of Alzheimer''s disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF)  = 0.015%), but that G330D is not (MAF  = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca²⁺ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca²⁺ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca²⁺ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.     

Sequence and Phylogenetic Analyses of 4 TMS Junctional Proteins of Animals: Connexins,Innexins, Claudins and Occludins

Connexins and probably innexins are the principal constituents of gap junctions, while claudins and occludins are principal tight junctional constituents. All have similar topologies with four alpha-helical transmembrane segments (TMSs), and all exhibit well-conserved extracytoplasmic cysteines that either are known to or potentially can form disulfide bridges. We have conducted sequence, topological and phylogenetic analyses of the proteins that comprise the connexin, innexin, claudin and occludin families. A multiple alignment of the sequences of each family was used to derive average hydropathy and similarity plots as well as phylogenetic trees. Analyses of the data generated led to the following evolutionary and functional suggestions: (1) In all four families, the most conserved regions of the proteins from each family are the four TMSs although the extracytoplasmic loops between TMSs 1 and 2, and TMSs 3 and 4 are usually well conserved. (2) The phylogenetic trees revealed sets of orthologues except for the innexins where phylogeny primarily reflects organismal source, probably due to a lack of relevant organismal sequence data. (3) The two halves of the connexins exhibit similarities suggesting that they were derived from a common origin by an internal gene duplication event. (4) Conserved cysteyl residues in the connexins and innexins may point to a similar extracellular structure involved in the docking of hemichannels to create intercellular communication channels. (5) We suggest a similar role in homomeric interactions for conserved extracellular residues in the claudins and occludins. The lack of sequence or motif similarity between the four different families indicates that, if they did evolve from a common ancestral gene, they have diverged considerably to fulfill separate, novel functions. We suggest that internal duplication was a general evolutionary strategy used to generate new families of channels and junctions with unique functions. These findings and suggestions should serve as guides for future studies concerning the structures, functions and evolutionary origins of junctional proteins.     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

CALHM1 and its polymorphism P86L differentially control Ca2+ homeostasis,mitogen‐activated protein kinase signaling,and cell vulnerability upon exposure to amyloid β

The mutated form of the Ca²⁺ channel CALHM1 (Ca²⁺ homeostasis modulator 1), P86L‐CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L‐CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca²⁺ removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L‐CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca²⁺ signal pathway; (ii) cell survival proteins ERK1/2 and Ca²⁺/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca²⁺ concentrations ([Ca²⁺]_n) and cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) on Ca²⁺ entry conditions, we observed that baseline [Ca²⁺]_n was higher in CALHM1 and P86L‐CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca²⁺]_c levels in HeLa cells overexpressing CALHM1 and P86L‐CALHM1 compared with control cells. Treatment with Aβ elicited a significant decrease in the cell survival proteins p‐ERK and p‐CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L‐CALHM1‐overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aβ, P86L‐CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro‐cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.     

Trafficking dynamics of glycosylated pannexin 1 proteins

Pannexins are mammalian orthologs of innexins and have a predicted topological folding pattern similar to that of connexins, except they are glycosylated. Rat pannexin 1 is glycosylated at N254 and this residue is important for plasma membrane targeting. Here we demonstrate that cell surface expression levels of the rat pannexin 1 N254Q mutant are rescued by coexpression with the wild-type protein. In paired Xenopus oocytes, the functional effect of this rescue is inconsequential; however, cell surface deglycosylation by PNGase F significantly enhanced functional gap junction formation. In mammalian cells, wild-type oligomers traffic at a slower rate than Myc-or tetracysteine domain-tagged versions, a behavior opposite to that of tagged connexins. The temporal differences of Panx1 trafficking correlate with spatial differences of intracellular localizations induced by Golgi blockage by Brefeldin-A or glycosylation prevention by tunicamycin. Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels.     

Genes, gene knockouts, and mutations in the analysis of gap junctions

Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.     

Gap junction gene and protein families: Connexins,innexins, and pannexins

Gap junction channels facilitate the intercellular exchange of ions and small molecules. While this process is critical to all multicellular organisms, the proteins that form gap junction channels are not conserved. Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. Interestingly, vertebrates and lower chordates contain innexin homologs, the pannexins, which also form channels, but rarely (if ever) make intercellular channels. While the connexin and the innexin/pannexin polypeptides do not share significant sequence similarity, all three of these protein families share a similar membrane topology and some similarities in quaternary structure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.     

The Pattern of Disulfide Linkages in the Extracellular Loop Regions of Connexin 32 Suggests a Model for the Docking Interface of Gap Junctions

Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of ~34–37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel β sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.     

Tryptophan scanning mutagenesis of the first transmembrane domain of the innexin Shaking-B(Lethal)

The channel proteins of gap junctions are encoded by two distinct gene families, connexins, which are exclusive to chordates, and innexins/pannexins, which are found throughout the animal kingdom. Although the relationship between the primary structure and function of the vertebrate connexins has been relatively well studied, there are, to our knowledge, no structure-function analyses of invertebrate innexins. In the first such study, we have used tryptophan scanning to probe the first transmembrane domain (M1) of the Drosophila innexin Shaking-B(Lethal), which is a component of rectifying electrical synapses in the Giant Fiber escape neural circuit. Tryptophan was substituted sequentially for 16 amino acids within M1 of Shaking-B(Lethal). Tryptophan insertion at every fourth residue (H27, T31, L35, and S39) disrupted gap junction function. The distribution of these sites is consistent with helical secondary structure and identifies the face of M1 involved in helix-helix interactions. Tryptophan substitution at several sites in M1 altered channel properties in a variety of ways. Changes in sensitivity to transjunctional voltage (Vj) were common and one mutation (S39W) induced sensitivity to transmembrane voltage (Vm). In addition, several mutations induced hemichannel activity. These changes are similar to those observed after substitutions within the transmembrane domains of connexins.     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels

Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the “slide helix” forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.     

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H⁺ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.     

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP₂), we found that PIP₂ increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP₂ or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr³⁷⁰ in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.     

A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk

Alzheimer's disease (AD) is a genetically heterogeneous disorder characterized by early hippocampal atrophy and cerebral amyloid-beta (Abeta) 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 Ca(2+) concentrations and Abeta levels. CALHM1 homomultimerizes, shares strong sequence similarities with the selectivity filter of the NMDA receptor, and generates a large Ca(2+) 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 x 10(-10)). We further found that the P86L polymorphism increases Abeta levels by interfering with CALHM1-mediated Ca(2+) permeability. We propose that CALHM1 encodes an essential component of a previously uncharacterized cerebral Ca(2+) channel that controls Abeta levels and susceptibility to late-onset AD.