Don-On Daniel Mak

MICU1 is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake That Regulates Cell Survival

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopusoocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [14C]methylamine (MA), had an apparent EC50 of 1.6 mm and a maximum uptake rate (V max) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na+ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H+ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC50 = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H+-coupled counter-transport mechanism.

Gain-of-Function Enhancement of IP3 Receptor Modal Gating by Familial Alzheimer’s Disease–Linked Presenilin Mutants in Human Cells and Mouse Neurons

By altering IP3 receptor gating, presenilin mutations associated with familial Alzheimer’s disease increase Ca2+ release from the endoplasmic reticulum. Opening the Calcium Floodgates? Alzheimer’s disease (AD), which is the most common cause of dementia, is a neurodegenerative disorder that affects some 5 million Americans. Although most cases of AD are sporadic, an early-onset form of familial AD (FAD) has been linked to mutations in the presenilins (PSs), transmembrane proteins localized to the endoplasmic reticulum (ER). Cheung et al. investigated the effects of wild-type and mutant forms of PS on inositol trisphosphate receptor (IP3R)–mediated Ca2+ release from the ER in various different cellular systems, including human lymphoblasts derived from individuals with FAD and cortical neurons from a mouse model of FAD. They found that FAD-linked PS mutants enhanced Ca2+ release by modulating IP3R channel gating through a gain-of-function mechanism, consistent with the autosomal-dominant inheritance of FAD. FAD-linked PS mutants, but not PS mutants associated with another form of dementia, shifted IP3R channel gating to a mode in which the probability that individual channels were open after stimulation was increased, leading to exaggerated Ca2+ signals. Familial Alzheimer’s disease (FAD) is caused by mutations in amyloid precursor protein or presenilins (PS1 and PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca2+) homeostasis by mechanisms proximal to and independent of amyloid production, although the molecular details are controversial. We found that several FAD-causing PS mutants enhance gating of the inositol trisphosphate receptor (IP3R) Ca2+ release channel by a gain-of-function effect that mirrored the genetics of FAD and was independent of secretase activity. In contrast, wild-type PS or PS mutants that cause frontotemporal dementia had no such effect. FAD-causing PS mutants altered the modes in which the IP3R channel gated. Recordings of endogenous IP3R in lymphoblasts derived from individuals with FAD or cortical neurons of asymptomatic PS1-AD mice revealed that they were more likely than IP3R in cells with wild-type PS to dwell in a high open-probability burst mode, resulting in enhanced Ca2+ signaling. These results indicate that exaggerated Ca2+ signaling through IP3R-PS interaction is a disease-specific and robust proximal mechanism in FAD.

ATP Regulation of Type 1 Inositol 1,4,5-Trisphosphate Receptor Channel Gating by Allosteric Tuning of Ca2+ Activation

Inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular Ca2+ by binding to its receptor (InsP3R), an endoplasmic reticulum-localized Ca2+ release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca2+ ([Ca2+] i ) dependence of single type 1 InsP3R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP] i ), but not the MgATP complex, activated gating of the InsP3-liganded InsP3R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca2+] i from 500 ± 50 nm in 0 [ATP] i to 29 ± 4 nm in 9.5 mm [ATP] i , with apparent ATP affinity = 0.27 ± 0.04 mm, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca2+activation. Thus, ATP enhances gating of the InsP3R by allosteric regulation of the Ca2+ sensitivity of the Ca2+ activation sites of the channel. By regulating the Ca2+-induced Ca2+ release properties of the InsP3R, ATP may play an important role in shaping cytoplasmic Ca2+ signals, possibly linking cell metabolic state to important Ca2+-dependent processes.

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.

Lack of Evidence for Presenilins as Endoplasmic Reticulum Ca2+ Leak Channels

Background: Presenilins (PS) were proposed to form endoplasmic reticulum Ca2+ channels, with their function disrupted in familial Alzheimer disease (FAD). Results: We found no evidence to support this but identified a technical artifact that led to the previous proposal. Conclusion: PS do not form endoplasmic reticulum Ca2+ channels. Significance: Exaggerated Ca2+ signaling in FAD is not caused by lack of PS Ca2+ channel function. Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca2+ ([Ca2+]i) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca2+]i signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca2+ leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca2+ release channel opening. To investigate this hypothesis, we employed multiple Ca2+ imaging protocols and indicators to directly measure ER Ca2+ dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca2+ leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca2+]i versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca2+ indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca2+ leak channels and highlight the need to use ER-targeted Ca2+ indicators when studying ER Ca2+ dynamics.

AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR

The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

Rapid ligand-regulated gating kinetics of single inositol 1,4,5-trisphosphate receptor Ca2+ release channels

The ubiquitous inositol 1,4,5‐trisphosphate receptor (InsP3R) intracellular Ca2+ release channel is engaged by thousands of plasma membrane receptors to generate Ca2+ signals in all cells. Understanding how complex Ca2+ signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP3 and Ca2+, which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP3R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch‐clamp configuration. The kinetics of channel activation and deactivation showed novel Ca2+ regulation and unexpected ligand cooperativity. The kinetics of Ca2+‐mediated channel inhibition showed the single‐channel bases for fundamental Ca2+ release events and Ca2+ release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca2+ signals.

Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels.

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a critical role in generation of complex Ca2+ signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP3R channels were consistently detected with regulation by cytoplasmic InsP3 and free Ca2+ concentrations ([Ca2+]i) very similar to that observed for vertebrate InsP3R. Long channel activity durations of the Sf9-InsP3R have now enabled identification of a novel aspect of InsP3R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP3R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P o) within each mode of 0.007 ± 0.002, 0.24 ± 0.03, and 0.85 ± 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca2+]i and [InsP3] does not substantially alter channel P o within a mode. Instead, [Ca2+]i and [InsP3] affect overall channel P o primarily by changing the relative probability of the channel being in each mode, especially the high and low P o modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP3R channel activity, with implications for the kinetics of Ca2+ release events in cells.