Adam P. Siebert

A Polymorphism in CALHM1 Influences Ca2+ Homeostasis, Aβ Levels, and Alzheimer's Disease Risk

Alzheimers 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.

Bitter and sweet taste receptors regulate human upper respiratory innate immunity

Bitter taste receptors (T2Rs) in the human airway detect harmful compounds, including secreted bacterial products. Here, using human primary sinonasal air-liquid interface cultures and tissue explants, we determined that activation of a subset of airway T2Rs expressed in nasal solitary chemosensory cells activates a calcium wave that propagates through gap junctions to the surrounding respiratory epithelial cells. The T2R-dependent calcium wave stimulated robust secretion of antimicrobial peptides into the mucus that was capable of killing a variety of respiratory pathogens. Furthermore, sweet taste receptor (T1R2/3) activation suppressed T2R-mediated antimicrobial peptide secretion, suggesting that T1R2/3-mediated inhibition of T2Rs prevents full antimicrobial peptide release during times of relative health. In contrast, during acute bacterial infection, T1R2/3 is likely deactivated in response to bacterial consumption of airway surface liquid glucose, alleviating T2R inhibition and resulting in antimicrobial peptide secretion. We found that patients with chronic rhinosinusitis have elevated glucose concentrations in their nasal secretions, and other reports have shown that patients with hyperglycemia likewise have elevated nasal glucose levels. These data suggest that increased glucose in respiratory secretions in pathologic states, such as chronic rhinosinusitis or hyperglycemia, promotes tonic activation of T1R2/3 and suppresses T2R-mediated innate defense. Furthermore, targeting T1R2/3-dependent suppression of T2Rs may have therapeutic potential for upper respiratory tract infections.

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability

Extracellular Ca2+ (Ca2+ o) plays important roles in physiology. Changes of Ca2+o concentration ([Ca2+]o) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca2+]o are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca2+ concentration ([Ca2+]i) in response to removal of Ca2+o and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca2+-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca2+]o. Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca2+]o with enhanced conductance and action potential firing and strongly elevated [Ca2+]i upon Ca2+o removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca2+ levels and plays a role in cortical neuronal excitability and Ca2+ homeostasis, particularly in response to lowering [Ca2+]o and its restoration to normal levels.

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins

Background: CALHM1 is an ion channel for which structural information is lacking. Results: CALHM1 has poor ion selectivity and a wide (∼14 Å) pore and is a hexamer, with monomers having four transmembrane domains with cytoplasmic termini. Conclusion: CALHM1 shares structural features with pannexins, connexins, and innexins. Significance: CALHMs, connexins, and pannexins/innexins are three structurally related protein families with shared and distinct functional properties. CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca2+ concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties.

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has InsP3-independent gating and disrupts intracellular Ca2+ homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) is a ubiquitous intracellular Ca2+ release channel that is vital to intracellular Ca2+ signaling. InsP3R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca2+ homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP3R1 and utilized a recombinant truncated form of the channel (capn-InsP3R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP3R1 revealed InsP3-independent gating and high open probability (Po) under optimal cytoplasmic Ca2+ concentration ([Ca2+]i) conditions. However, some [Ca2+]i regulation of the cleaved channel remained, with a lower Po in suboptimal and inhibitory [Ca2+]i. Expression of capn-InsP3R1 in N2a cells reduced the Ca2+ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca2+ loading compared with control cells expressing full-length InsP3R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP3R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP3R1 generates a dysregulated channel that disrupts cellular Ca2+ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP3R1 in a clinically relevant injury model, suggesting that Ca2+ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor impairs ER Ca2+ buffering and causes neurodegeneration in primary cortical neurons

Disruption of neuronal Ca2+ homeostasis plays a well‐established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5‐trisphosphate receptor (InsP3R1), a Ca2+ release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca2+ signaling and neurodegeneration in disease states. However, the specific effects of InsP3R1 proteolysis on neuronal Ca2+ homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain‐mediated InsP3R1 proteolysis on neuronal Ca2+ signaling and survival using adeno‐associated viruses to express a recombinant cleaved form of the channel (capn‐InsP3R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn‐InsP3R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca2+ concentration ([Ca2+]i), increased [Ca2+]i response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP3R1 proteolysis disrupts neuronal Ca2+ homeostasis, and potentially acts as a feed‐forward pathway to initiate or execute neuronal death.

Redox-Regulated Heterogeneous Thresholds for Ligand Recruitment among InsP3R Ca2+-Release Channels

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.

Denatonium-induced sinonasal bacterial killing may play a role in chronic rhinosinusitis outcomes: Denatonium-responsiveness and CRS outcomes

Sinonasal bitter taste receptors (T2Rs) contribute to upper airway innate immunity and correlate with chronic rhinosinusitis (CRS) clinical outcomes. A subset of T2Rs expressed on sinonasal solitary chemosensory cells (SCCs) are activated by denatonium, resulting in a calcium‐mediated secretion of bactericidal antimicrobial peptides (AMPs) in neighboring ciliated epithelial cells. We hypothesized that there is patient variability in the amount of bacterial killing induced by different concentrations of denatonium and that the differences correlate with CRS clinical outcomes.

A novel Calcium Channel Induced by CALHM1 Expression Detects Levels of Extracellular Calcium

CALHM1 was identified as an integral membrane protein expressed in the hippocampus and linked to late onset Alzheimers disease. Its heterologous expression was shown to modify membrane permeabilities, but the biophysical properties of induced currents and its physiological roles have not been defined. Expression of CALHM1 induces a novel voltage-gated conductance, activated upon depolarization and deactivated by hyperpolarization in a physiological solution (2 mM Ca2+ & 1 mM Mg2+). In addition, CALHM1-induced currents can be activated by lowering extracellular Ca2+ concentration ([Ca2+]o) even at the resting potentials in Xenopus oocytes and mammalian cell lines. Extracellular calcium (Ca2+o) does not affect the single-channel conductance (∼ 25 pS) in a wide range of voltages, and instantaneous I-V relations remain linear in presence or absence of Ca2+o. Ca2+o regulation involves modulation of a weakly intrinsic voltage-dependent gate rather than voltage-dependent pore block with a high selectivity over Mg2+. The CALHM1-induced permeability is non-selective among monovalent cations with substantial Cl- permeability (PNa/PK/PCl=1:1.17:0.56), with relatively high Ca2+ selectivity (PCa/PNa=10), indicating that Ca2+o may regulate cytosolic Ca2+ concentration and membrane potential through opening of CALHM1 induced channels. Gd3+ and ruthenium red inhibited the currents elicited by lowering Ca2+o, whereas blockers of connexins, NMDA receptors and other voltage-gated channels were without effect. Lowering Ca2+o in the solution containing 1 mM Mg2+ to block TRPM7 channels strongly depolarized and excited cultured cortical neurons from WT but not CALHM1 KO mice. Together these results suggest that CALHM1 expression induces a novel voltage- and Ca2+o-regulated Ca2+ permeability that regulates neuronal excitability. Insights into the molecular basis of the CALHM1-induced currents and its gating mechanisms may provide clues regarding the physiological roles of CALHM1 in physiological and pathological conditions, and to facilitate therapeutic interventions in Alzheimers disease.