Neurosci Lett

Recently, the P86L alteration in CALHM1 (calcium homeostasis modulator-1) was reported to be associated with Alzheimer's disease (AD). Moreover, the risk allele increased amyloid-beta (A beta) levels in conditioned media from cultured cells. Therefore, we hypothesized that CALHM1 P86L may modulate A beta or tau levels in cerebrospinal fluid (CSF). Nearly 200 individuals with AD or other cognitive disorders were included for CSF analysis and CALHM1 genotyping. No significant differences in CSF levels of A beta 42, tau or phospho-tau were found across the various CALHM1 genotypes. In conclusion, we found no evidence that CALHM1 P86L is associated with altered CSF levels of the investigated AD biomarkers

When, where and how? Focus on neuronal calcium dysfunctions in Alzheimer's Disease.

Alzheimer\u2019s disease (AD), since its characterization as a precise form of dementia with its own pathological hallmarks, has captured scientists\u2019 attention because of its complexity. The last 30 years have been filled with discoveries regarding the elusive aetiology of this disease and, thanks to advances in molecular biology and live imaging techniques, we now know that an important role is played by calcium (Ca2+). Ca2+, as ubiquitous second messenger, regulates a vast variety of cellular processes, from neuronal excitation and communication, to muscle fibre contraction and hormone secretion, with its action spanning a temporal scale that goes from microseconds to hours. It is therefore very challenging to conceive a single hypothesis that can integrate the numerous findings on this issue with those coming from the classical fields of AD research such as amyloid-beta (A) and tau pathology. In this contribution, we will focus our attention on the Ca2+ hypothesis of AD, dissecting it, as much as possible, in its subcellular localization, where the Ca2+ signal meets its specificity. We will also follow the temporal evolution of the Ca2+ hypothesis, providing some of the most updated discoveries. Whenever possible, we will link the findings regarding Ca2+ dysfunction to the other players involved in AD pathogenesis, hoping to provide a crossover body of evidence, useful to amplify the knowledge that will lead towards the discovery of an effective therapy

Calcium in the initiation, progression and as an effector of Alzheimer's disease pathology.

The cause(s) of sporadic Alzheimer's disease (sAD) are complex and currently poorly understood. They likely result from a combination of genetic, environmental, proteomic and lipidomic factors that crucially occur only in the aged brain. Age-related changes in calcium levels and dynamics have the potential to increase the production and accumulation of both amyloid-beta peptide (Abeta) and tau pathologies in the AD brain, although these two pathologies themselves can induce calcium dyshomeostasis, particularly at synaptic membranes. This review discuses the evidence for a role for calcium dyshomeostasis in the initiation of pathology, as well as the evidence for these pathologies themselves disrupting normal calcium homeostasis, which lead to synaptic and neuronal dysfunction, synaptotoxicity and neuronal loss, underlying the dementia associated with the disease

Molecular and pharmacological modulation of CALHM1 promote neuroprotection against oxygen and glucose deprivation in a model of hippocampal slices

Calcium homeostasis modulator 1 (CALHM1) is a calcium channel involved in the regulation of cytosolic Ca2+ levels. From a physiological point of view, the open state of CALHM1 depends not only on voltage but also on the extracellular concentration of calcium ([Ca2+]) ions. At low [Ca2+]e or depolarization, the channel is opened, allowing Ca2+ influx; however, high extracellular [Ca2+]e or hyperpolarization promote its resting state. The unique Ca2+ permeation of CALHM1 relates to the molecular events that take place in brain ischemia, such as depolarization and extracellular changes in [Ca2+]e, particularly during the reperfusion phase after the ischemic insult. In this study, we attempted to understand its role in an in vitro model of ischemia, namely oxygen and glucose deprivation, followed by reoxygenation (OGD/Reox). To this end, hippocampal slices from wild-type Calhm1+/+, Calhm1+/-, and Calhm1-/- mice were subjected to OGD/Reox. Our results point out to a neuroprotective effect when CALHM1 is partially or totally absent. Pharmacological manipulation of CALHM1 with CGP37157 reduced cell death in Calhm1+/+ slices but not in that of Calhm1-/- mice after exposure to the OGD/Reox protocol. This ionic protection was also verified by measuring reactive oxygen species production upon OGD/Reox in Calhm1+/+ and Calhm1-/- mice, resulting in a downregulation of ROS production in Calhm1-/- hippocampal slices and increased expression of HIF-1α. Taken together, we can conclude that genetic or pharmacological inhibition of CALHM1 results in a neuroprotective effect against ischemia, due to an attenuation of the neuronal calcium overload and downregulation of oxygen reactive species productionThis research was funded by the Spanish Ministry of Science, Innovation and Universities Ref RTI2018-095793-B-I00 and Comunidad Autónoma de Madrid Ref. B2017/BMD-3827 to MGL. ETN PURINESDX Research and Innovation Agreement Nº 766124. Program under the Marie Sklodowska-Curie and Proyectos Santander-Universidad Autónoma de Madrid 2017, to MFC

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 (Ab) 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 Ab 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 Ab. 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 Ab affected [Ca2+]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Ab 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 Ab, 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.This work was partly supported by the following grants: Ministerio de Economía y Competitividad, FPU Program, Refs. AP2009/0343 (AJMO) and AP2010/1219 (IB). ARN: FIS PI10/01426. MGL: Ministerio de Economía y Competitividad, Ref. SAF2012-23332. MFCA: Consolidación de grupos de investigación UAM-CAM 1004040047. We also thank Fundación Teófilo Hernando, Madrid, Spain, for their continued suppor

CALHM1-Mediated ATP Release and Ciliary Beat Frequency Modulation in Nasal Epithelial Cells

Mechanical stimulation of airway epithelial cells causes apical release of ATP, which increases ciliary beat frequency (CBF) and speeds up mucociliary clearance. The mechanisms responsible for this ATP release are poorly understood. CALHM1, a transmembrane protein with shared structural features to connexins and pannexins, has been implicated in ATP release from taste buds, but it has not been evaluated for a functional role in the airway. In the present study, Calhm1 knockout, Panx1 knockout, and wild-type mouse nasal septal epithelial cells were grown at an air-liquid interface (ALI) and subjected to light mechanical stimulation from an air puff. Apical ATP release was attenuated in Calhm1 knockout cultures following mechanical stimulation at a pressure of 55 mmHg for 50 milliseconds (p \u3c 0.05). Addition of carbenoxolone, a PANX1 channel blocker, completely abolished ATP release in Calhm1 knockout cultures but not in wild type or Panx1 knockout cultures. An increase in CBF was observed in wild-type ALIs following mechanical stimulation, and this increase was significantly lower (p \u3c 0.01) in Calhm1 knockout cultures. These results demonstrate that CALHM1 plays a newly defined role, complementary to PANX1, in ATP release and downstream CBF modulation following a mechanical stimulus in airway epithelial cells. © 2017 The Author(s)

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes. Ma et al. identify a CALHM1/CALHM3 hetero-hexameric ion channel as the mechanism by which type II taste bud cells release ATP as a neurotransmitter to gustatory neurons in response to GPCR-mediated tastes, including sweet, bitter, and umami substances. © 2018 Elsevier Inc

Structural and Functional Studies of Large-Pore Channels of the CALHM Family

A cell relies on proteins, termed ion channels, that facilitate the passage of ions across its membranes. These channels contain pores that activate in response to certain stimuli, allowing ion flow in their open state. Previous research has predominantly focused on ion channels with narrow and selective pores, which permit only certain types of inorganic ions to cross the membrane. However, there is increasing interest in proteins that form channels with wider pores that facilitate the permeation of larger substrates. These large-pore channels play various biological roles by allowing signaling molecules such as ATP to cross the membrane. One family of large-pore channels is the recently discovered calcium homeostasis modulators (CALHM) family, which contains six paralogs in humans. CALHM1 has been shown to form ion channels that are activated by voltage and the decrease of extracellular calcium. It pairs with CALHM3 to form heteromeric channels with improved activation properties. CALHM1 and 3 have been found in primate taste buds, and their role in perceiving sweet, bitter, and umami tastes through ATP efflux and subsequent purinergic signaling has been demonstrated. Despite these breakthroughs, the mechanisms underlying ion permeation and gating of CALHM channels, and the function of other family members have remained elusive. In this thesis project, I aimed to study the functional and structural features of human CALHM proteins, with a particular focus on uncharacterized members of the family. To achieve this goal, I have identified biochemically stable constructs of CALHM2, CALHM4, and CALHM6 and used cryo-electron microscopy to characterize their detailed three-dimensional structures. These findings provided the first structures of CALHM4 and CALHM6, revealing their unique features to assemble as large channels composed of ten or eleven subunits with a central pore. Strikingly, I observed two different conformations between the homologues that significantly impacted pore size and geometry, potentially suggesting a gating mechanism. In one conformation, the pore-lining α-helix 1 runs parallel to the pore axis, creating a wide-open cylindrical pore, while in the other conformation, the same elements lifts to form a conical pore, which narrows towards the cytoplasm. Additionally, the CALHM4 structure exhibited extra bilayer-like density inside the pore, which likely either originates from detergents used in the purification or co-purified lipids, indicating a potential role of lipids in the regulation of channel activity. Although the physiological relevance of observed features remains unclear, the described work has provided valuable insight into the general architecture and potential activation mechanisms of the CALHM channels. Complementary functional studies of CALHM2, CALHM4, and CALHM6 by electrophysiology and in vitro transport assays did not show any activity under conditions that activate CALHM1, suggesting a distinct mechanism of activation. Transcript analysis demonstrated that all three homologues are highly enriched in the placenta, prompting the investigation of mutual interactions in vitro. I could show that CALHM2 and CALHM4 interact to form heteromeric CALHM channels, similar to the previously reported CALHM1/3 heteromers. However, functional studies did not show any activity in cells co-expressing CALHM2 and CALHM4, in contrast to the altered phenotype of 2 CALHM1/3 heteromers and similar to the lack of activity that was previously observed in homomeric CALHM2, 4 and 6 channels. Despite the observed lack of activity, I pursued the structural characterization of heteromeric CALHM2/4 complexes in the hope to uncover structural features that facilitate the comprehension of CALHM function. To overcome the challenge of identifying structurally similar subunits in a heteromeric channel, I have generated specific sybodies to each of the two homologues to facilitate their distinction during reconstruction. Before structure determination of heteromeric channel complexes, I first characterized the homomeric CALHM paralogs in complex with their respective sybodies, which identified their respective binding sites. These efforts also yielded a structure of CALHM2, which was prevented in case of the apo-protein due to the preferred channel orientation on cryo-EM grids. CALHM2, like CALHM4 and 6, form large channels and its pore conformation is similar as observed for CALHM6. The structure of CALHM2/4 complexes revealed heteromeric channels with broad distribution of oligomeric states, ranging from decamers to dodecamers, with varying subunit stoichiometries. The high-resolution structure of a predominant population showed a heteromeric channel with an excess of CALHM2 subunits that are arranged in a cluster and a group of two to three CALHM4 subunits. While most subunits reside in the preferred conformation defined in homomeric structures, the conformation of a CALHM2 subunit, located in the neighborhood of CALHM4, differed in conformation, aiding the understanding on the conversion of states. Intriguingly, similar to the CALHM4 structure, extra density was observed in a section of the pore close to the subunits residing in cylindrical conformations. This further supports the notion that the pore-lining α-helix and lipids may play a crucial role in the gating of CALHM channels, which likely employ complex regulatory mechanisms that may be distinct for different members of the family

Opening a “Wide” Window onto Taste Signal Transmission

Taste bud cells for sweet, umami, and bitter transmit sensory signals without a synapse. A study by Ma et al. (2018) finds a key ATP-permeable pore-forming subunit required for rapid neurotransmission from the tongue to secondary taste neurons