Horia Vais

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

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.

Apoptosis regulation by Bcl-xL modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating

Members of the Bcl-2 family of proteins regulate apoptosis, with some of their physiological effects mediated by their modulation of endoplasmic reticulum (ER) Ca2+ homeostasis. Antiapoptotic Bcl-xL binds to the inositol trisphosphate receptor (InsP3R) Ca2+ release channel to enhance Ca2+- and InsP3-dependent regulation of channel gating, resulting in reduced ER [Ca2+], increased oscillations of cytoplasmic Ca2+ concentration ([Ca2+]i), and apoptosis resistance. However, it is controversial which InsP3R isoforms mediate these effects and whether reduced ER [Ca2+] or enhanced [Ca2+]i signaling is most relevant for apoptosis protection. DT40 cell lines engineered to express each of the three mammalian InsP3R isoforms individually displayed enhanced apoptosis sensitivity compared with cells lacking InsP3R. In contrast, coexpression of each isoform with Bcl-xL conferred enhanced apoptosis resistance. In single-channel recordings of channel gating in native ER membranes, Bcl-xL increased the apparent sensitivity of all three InsP3R isoforms to subsaturating levels of InsP3. Expression of Bcl-xL reduced ER [Ca2+] in type 3 but not type 1 or 2 InsP3R-expressing cells. In contrast, Bcl-xL enhanced spontaneous [Ca2+]i signaling in all three InsP3R isoform-expressing cell lines. These results demonstrate a redundancy among InsP3R isoforms in their ability to sensitize cells to apoptotic insults and to interact with Bcl-xL to modulate their activities that result in enhanced apoptosis resistance. Furthermore, these data suggest that modulation of ER [Ca2+] is not a specific requirement for ER-dependent antiapoptotic effects of Bcl-xL. Rather, apoptosis protection is conferred by enhanced spontaneous [Ca2+]i signaling by Bcl-xL interaction with all isoforms of the InsP3R.

EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter.

The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.

Graded recruitment and inactivation of single InsP3 receptor Ca2+-release channels: implications for quartal Ca2+release

Modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) by receptor‐mediated generation of inositol 1,4,5‐trisphosphate (InsP3) and activation of its receptor (InsP3R), a Ca2+‐release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP3‐mediated signalling is the graded release of Ca2+ in response to incremental levels of stimuli. Ca2+ release has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3]. Many schemes have been proposed to account for quantal Ca2+ release, including the presence of heterogeneous channels and Ca2+ stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP3R channel can account for quantal Ca2+ release. Patch‐clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting functional endogenous InsP3R channels, enabling InsP3‐induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number of activated channels in the membrane patch (NA) to be accurately quantified. InsP3‐activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca2+]i and suboptimal [InsP3]. Unexpectedly, NA was found to be a graded function of both [Ca2+]i and [InsP3]. A qualitative model involving Ca2+‐induced InsP3R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP3R channels, providing a mechanism for graded Ca2+ release that is intrinsic to the InsP3R Ca2+ release channel itself.

MCUR1, CCDC90A, Is a Regulator of the Mitochondrial Calcium Uniporter

MCU is the pore-forming subunit of the mitochondrial inner membrane Ca2+ uniporter ion channel that mediates Ca2+ uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca2+ signaling. We previously identified MCUR1 (Mitochondrial Calcium Uniporter Regulator 1) as an important regulator of MCU activity and showed that MCUR1 biochemically interacted with MCU (Mallilankaraman et al., 2012). MCUR1 regulated MCU-dependent mitochondrial Ca2+ uptake driven by the inner membrane voltage (ψm) generated by the electron transport chain, and MCUR1 knockdown abrogated Ca2+ uptake by mitochondria in intact and permeabilized cells, and disrupted oxidative phosphorylation (OXPHOS).

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.

Unitary Ca2+ current through recombinant type 3 InsP3 receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca2+ into the cytoplasm upon binding InsP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open InsP3R channel determines the amount of Ca2+ released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca2+ signals generated in response to extracellular stimuli. Despite its significance, iCa for InsP3R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of iCa through an InsP3R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP3R channels. Within physiological ranges of free Ca2+ concentrations in the ER lumen ([Ca2+]ER), free cytoplasmic [Ca2+] ([Ca2+]i), and symmetric free [Mg2+] ([Mg2+]f), the iCa–[Ca2+]ER relation was linear, with no detectable dependence on [Mg2+]f. iCa was 0.15 ± 0.01 pA for a filled ER store with 500 µM [Ca2+]ER. The iCa–[Ca2+]ER relation suggests that Ca2+ released by an InsP3R channel raises [Ca2+]i near the open channel to ∼13–70 µM, depending on [Ca2+]ER. These measurements have implications for the activities of nearby InsP3-liganded InsP3R channels, and they confirm that Ca2+ released by an open InsP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl− Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl− and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl− secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca2+ from the endoplasmic reticulum (ER), lowering [Ca2+] in the ER and thereby activating the Ca2+-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca2+] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl− current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca2+ buffer that lowers [Ca2+] in the ER similar to the effect of 3O-C12 also increased cAMP and ICl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl− and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca2+] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl− and fluid secretion.

Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

Significance Changes in Ca2+ concentration in the cell play important roles in cell life and death decisions. Antiapoptotic Bcl-2 family proteins help protect cells from dying by interacting with proteins at mitochondria and endoplasmic reticulum. At the endoplasmic reticulum, antiapoptotic Bcl-2 proteins interact with InsP3R Ca2+ channels that release Ca2+ into the cytoplasm. However, it is controversial how they interact with the InsP3R, as well as the functional consequences of the interactions. We found that antiapoptotic Bcl-xL interacts with InsP3Rs by unique mechanisms that change the activity of the channel depending on its concentration. We also found that disrupting these interactions diminishes cell viability. Our results provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP3R. Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP3R) Ca2+ release channels in the endoplasmic reticulum to modulate Ca2+ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-xL activates single InsP3R channels with a biphasic concentration dependence. The Bcl-xL Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-xL activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP3R that play critical roles in Ca2+ signaling and cell viability.