Don-on Mak - Academia.edu (original) (raw)

Papers by Don-on Mak

PLOS Computational Biology, 2015

Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-t... more Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP 3) receptor (IP 3 R) Ca 2+ release channels resulting in enhanced IP 3 R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP 3 R activity is considered to be the main reason behind the upregulation of intracellular Ca 2+ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca 2+ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP 3 R channel activity records obtained under optimal Ca 2+ and multiple IP 3 concentrations to gain deeper insights into the enhancement of IP 3 R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP 3 R in FAD-causing mutant PSexpressing cells exhibits significantly longer mean lifetime for the H mode and shorter lifetime for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP 3 R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP 3 and Ca 2+ concentrations and quantify the sensitivity of IP 3 R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP 3 and Ca 2+ and that very small amount of IP 3 is required to stimulate IP 3 R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP 3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP 3 R in the H mode leads to the observed higher frequency of local Ca 2+ signals, which can account for the more frequent global Ca 2+ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca 2+ signals in cells expressing FAD-causing mutant PS.

Proceedings of the National Academy of Sciences, 2002

The inositol trisphosphate (Ins P 3 ) receptor (Ins P 3 R) is a ubiquitously expressed intracellu... more The inositol trisphosphate (Ins P 3 ) receptor (Ins P 3 R) is a ubiquitously expressed intracellular Ca 2+ channel that mediates complex cytoplasmic Ca 2+ signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the Ins P 3 R channel is normally thought to require binding of Ins P 3 derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca 2+ -binding proteins as high-affinity protein agonists of the Ins P 3 R, which bind to the channel and activate gating in the absence of Ins P 3 . CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the Ins P 3 -binding region of all three Ins P 3 R channel isoforms with high affinity (K a ≈ 25 nM) in a Ca 2+ -dependent manner (K a ≈ 1 μM). Binding activates single-channel gating as efficaciously as Ins P 3 , dependent on functional EF-hands in CaBP. In contras...

The inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R), a Ca 2 �-release channel localized ... more The inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R), a Ca 2 �-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca 2 � signals in many cell types. Three InsP 3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP 3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca 2 � concentrations ([Ca 2 � ] i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca 2 � ] in mediating high [Ca 2 � ] i inhibition of InsP 3R gating by studying single endogenous type 1 InsP 3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca 2 �...

The inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) is an endoplasmic reticulum–localize... more The inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) is an endoplasmic reticulum–localized Ca 2 �-release channel that controls complex cytoplasmic Ca 2 � signaling in many cell types. At least three InsP 3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP 3R (r-InsP 3R-3) was expressed in Xenopus oocytes, and singlechannel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP 3R-3 exhibited a biphasic dependence on cytoplasmic free Ca 2 � concentration ([Ca 2 � ] i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP 3R-3 gating was inhibited by high [Ca 2 � ] i with features similar to those of the endogenous Xenopus type 1 InsP 3R (X-InsP 3R-1). Ca 2 � inhibition of channel gating had an ...

Biophysical Journal

The ability of Kþ channel agonists or 'openers' to dampen cellular electrical excitability makes ... more The ability of Kþ channel agonists or 'openers' to dampen cellular electrical excitability makes them promising pharmacological tools for the treatment of many neurological and cardiovascular disorders such as epilepsy, pain, inflammation, arrhythmias, hypertension, and even erectile dysfunction. Consequently, a large number of Kþ channel openers have been identified. However, in most cases their binding sites and mechanisms of action are unknown thereby preventing the rational design of more effective or selective compounds. Different classes of Kþ channels have been evolutionarily tuned for their activation by divers biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. These various mechanisms, however, finally converge on one of two gates in Kþ channels, a lower gate at the cytoplasmic pore entrance and an upper gate at the selectivity filter (SF). Here we report on a new class of negatively-charged activators (NCAs) that universally target Kþ channels operated at the SF gate including many K2P channels, voltage-gated hERG channels and Ca2þ-activated BK-type Kþ channels. Functional analysis combined with crystallographic studies and molecular dynamics simulations revealed a conserved NCA effector mechanism that involves electrostatic coordination of Kþ ions beneath the SF and stabilization of the open SF conformation. These results disclose an unrecognized polypharmacology for many known Kþ channel openers and an unique drug interaction principle, highlight a SF gating machinery conserved across Kþ channel families and advance our general understanding of drug action in ion channels.

Cell Calcium

Intracellular accumulation of oligomeric forms of β amyloid (Aβ) are now believed to play a key r... more Intracellular accumulation of oligomeric forms of β amyloid (Aβ) are now believed to play a key role in the earliest phase of Alzheimer's disease (AD) as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aβ oligomers. However, little is known about the downstream effects of the resulting Ca2+ rise on the many intracellular Ca2+-dependent pathways. Here we use multiscale modeling in conjunction with patch-clamp electrophysiology of single inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and fluorescence imaging of whole-cell Ca2+ response, induced by exogenously applied intracellular Aβ42 oligomers to show that Aβ42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the endoplasmic reticulum by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and H2O2. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aβ42 cytotoxicity by EGTA in our live cells experiments.

Biophysical journal, Jan 3, 2018

Experimental records of single molecules or ion channels from fluorescence microscopy and patch-c... more Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. Moreover, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and likely to hinder the processing of often very large data sets. We introduce a maximal likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca release events called puffs arising from clusters of channels, and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and ba...

Biophysical Journal, 2017

the maximum level, further oxidation of RyR2 did not enhance SR Ca leak. These results indicate t... more the maximum level, further oxidation of RyR2 did not enhance SR Ca leak. These results indicate that the XL is a strong regulator of RyR2 function. We examined if RyR2 XL occurs during cardiac pathologies associated with oxidative stress. Ventricular myocytes isolated from rabbit failing hearts were in the state of oxidative stress since they exhibited an increase in reactive oxygen species level and the overall protein oxidation. These myocytes were also characterized by a significant level of RyR2 XL. Measurements of [Ca] within the SR revealed that SR Ca leak is significantly increased in HF. Moreover, the frequency-dependent inotropy was largely blunted in HF due to the inability of the SR to maintain Ca load at high pacing rates. Since SR Ca load is determined by a balance between SR Ca uptake and leak, the decline of the frequency-dependent inotropy in HF can be mediated by the XL-dependent SR Ca leak. Thus, we studied if RyR2 XL alone can cause the blunted frequency-dependent inotropy in control myocytes. When RyR2 XL was induced in control myocytes, the frequency-dependent inotropy was significantly suppressed. At the same time, oxidative stress did not affect SR Ca uptake, suggesting that SR Ca leak induced by RyR2 XL plays a key role in the blunted frequency-dependent inotropy during oxidative stress.

Cell Calcium, 2016

Ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (InsP 3 R) are the predomina... more Ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (InsP 3 R) are the predominant intracellular Ca 2+ release channels in cells, localized predominately in the sarco-and endoplasmic reticulum (ER), the major intracellular Ca 2+ reservoirs, and regulating diverse cellular processes. The two channel families are each comprised of three distinct genes with widespread expression throughout the body. RyR1 plays an important role in skeletal muscle where, through an interaction with voltage-gated Ca 2+ channels (VGCC) in the transverse tubule membrane, it mediates excitation-contraction coupling. RyR2 is expressed in the heart, where it is activated during each heartbeat by Ca 2+ influx through plasma membrane VGCC by the process of Ca 2+-induced Ca 2+ release. RyR3 is expressed in muscle in some species, as well as in the brain. The type 1 InsP 3 R (InsP 3 R-1) is highly expressed in cerebellar Purkinje neurons, although it and the other two InsP 3 R isoforms have widespread expression patterns. All three InsP 3 Rs are ligand gated ion channels, activated by binding to InsP 3 generated by engagement of plasma membrane receptors coupled to the activation of phospholipases C [1]. These families of ion channels are among the largest known. As homo-tetramers, RyRs have a molecular mass ~ 2.3 MDa. InsP 3 R homo-and hetero-tetramers are smaller, with a molecular mass ~1.2 MDa.

Cell Reports, 2016

Highlights d The inner membrane mitochondrial MCU ion channel mediates Ca 2+ uptake into the matr... more Highlights d The inner membrane mitochondrial MCU ion channel mediates Ca 2+ uptake into the matrix d MCU channel activity is governed by matrix Ca 2+ concentration through EMRE d EMRE-dependent regulation requires MICU1, MICU2, and cytoplasmic Ca 2+ d EMRE couples Ca 2+ sensors on both sides of the inner membrane to regulate MCU

Journal of Biological Chemistry

The Journal of Cell Biology

Current topics in membranes, 2010

Publisher Summary The activity of the inositol 1,4,5-trisphosphate receptor (IP 3 R) Ca 2+ releas... more Publisher Summary The activity of the inositol 1,4,5-trisphosphate receptor (IP 3 R) Ca 2+ release channel is intricately regulated by a multitude of ligands. The most important ligands regulating IP 3 R channel activity are IP 3 and Ca 2+ . Generally, cytoplasmic Ca 2+ regulates steady-state IP 3 R channel gating with a biphasic concentration dependence—Ca 2+ at low concentrations activates the channel and increases its open probability, whereas at higher concentrations, Ca 2+ inhibits the channel. Ca 2+ modulates channel activity by binding to several apparently distinct sites that regulate Ca 2+ activation, IP 3 -dependent Ca 2+ inhibition, IP 3 -independent Ca 2+ inhibition, channel inactivation, and channel recruitment. A separate Ca 2+ -binding site appears to regulate the functionality of the IP 3 -independent Ca 2+ inhibition site. Quantitative and qualitative molecular models can account for much of the steady-state and kinetic behaviors of IP 3 R channel gating regulation by cytoplasmic Ca 2+ . The identification of modal gating has revealed that Ca 2+ modulates IP 3 R channel activity primary by regulating its propensity to gate in particular modes. Ca 2+ regulation of the channel is impinged on by other channel regulators, including IP 3 and ATP, as allosteric modulators.

The Journal of biological chemistry, Jan 27, 2001

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determin... more We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to w...

Cell calcium, Jan 18, 2014

As an intracellular Ca(2+) release channel at the endoplasmic reticulum membrane, the ubiquitous ... more As an intracellular Ca(2+) release channel at the endoplasmic reticulum membrane, the ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a crucial role in the generation, propagation and regulation of intracellular Ca(2+) signals that regulate numerous physiological and pathophysiological processes. This review provides a concise account of the fundamental single-channel properties of the InsP3R channel: its conductance properties and its regulation by InsP3 and Ca(2+), its physiological ligands, studied using nuclear patch clamp electrophysiology.

Cold Spring Harbor protocols, 2013

Patch-clamping the outer or inner nuclear membrane of isolated nuclei is very similar to patch-cl... more Patch-clamping the outer or inner nuclear membrane of isolated nuclei is very similar to patch-clamping the plasma membrane of isolated cells. This protocol describes in detail all the steps required to successfully obtain nuclear membrane patches, in various configurations, from both the outer and inner nuclear membranes of isolated nuclei.

Calcium Signaling, Second Edition, 2005

Proceedings of the National Academy of Sciences of the United States of America, 2006

Anesthesiology, 2014

Background: Pharmacological evidence suggests that inhalational general anesthetics induce neurod... more Background: Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP3R) Ca2+-release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics. Methods: Using single-channel patch clamp electrophysiology, the authors examined the gating of rat recombinant type 3 InsP3R (InsP3R-3) Ca2+-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist inositol 1,4,5-trisphosphate (InsP3). The authors also examined the effects of isoflurane on InsP3R-mediated Ca2+ release from the endoplasmic reticulum and changes in intracellular Ca2+ concentration ([Ca2+]i). Results: Clinically relevant concentrations (approximately 1 minimal alveolar concentration) of the commonly...