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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 Ca(2+) homeostasis as a direct consequence of the intracellular Aβ oligomers. However, little is known about the downstream effects of the resulting Ca(2+) rise on the many intracellular Ca(2+)-dependent pathways. Here we use multiscale modeling in conjunction with patchclamp electrophysiology of single inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and fluorescence imaging of whole-cell Ca(2+) response, induced by exogenously applied intracellular Aβ(42) oligomers to show that Aβ(42) inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP(3)R, which is then extended to build a model for the whole-cell Ca(2+) signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca(2+) release from the endoplasmic reticulum by intracellular Aβ(42) oligomers through G-protein-mediated stimulation of IP(3) production. The estimated IP(3) concentration as a function of intracellular Aβ(42) content together with the whole-cell model allows us to show that Aβ(42) oligomers impair mitochondrial function through pathological Ca(2+) uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and H(2)O(2). We further show that mitochondrial function can be restored by the addition of Ca(2+) buffer EGTA, in accordance with the observed abrogation of Aβ(42) cytotoxicity by EGTA in our live cells experiments.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.

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. More-over, 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 hinder the processing of often very large data-sets. We introduce a maximum likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ 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 back-ground signal can all be optimized using the expectation-maximization (EM) algorithm. We implement our algorithm following the Baum-Welch approach to EM in the portable java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters, and exporting a range of statistics such as the mean open probabilities, mean open times, mean close times, and dwell time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click are provided.

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 used general anesthetic, isoflurane, activated InsP3R-3 channels with open probability similar to channels activated by 1 µM InsP3 (Po ≈ 0.2). This isoflurane modulation of InsP3R-3 Po depended biphasically on [Ca2+]i. Combination of isoflurane with subsaturating levels of InsP3 in patch pipettes resulted in at least two-fold augmentations of InsP3R-3 channel Po compared with InsP3 alone. These effects were not noted in the presence of saturating [InsP3]. Application of isoflurane to DT40 cells resulted in a 30% amplification of InsP3R-mediated [Ca2+]i oscillations, whereas InsP3-induced increase in [Ca2+]i and cleaved caspase-3 activity were enhanced by approximately 2.5-fold. Conclusion: These results suggest that the InsP3R may be a direct molecular target of isoflurane and plays a role in the mechanisms of anesthetic-mediated pharmacological or neurotoxic effects.

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) Ca(2+) release channel plays a central role in the generation and modulation of intracellular Ca(2+) signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP(3), free Ca(2+), free ATP(4-)) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP(3)R channel activity by free Ca(2+) in the ER lumen ([Ca(2+)](ER)) remains poorly understood because of limitations of Ca(2+) flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca(2+)](ER) on homotetrameric rat type 3 InsP(3)R channel activity. In optimal [Ca(2+)](i) and subsaturating [InsP(3)], jumps of [Ca(2+)](ER) from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP(3) but restored when [Ca(2+)](ER) was raised to 1.1 mM. In suboptimal [Ca(2+)](i), jumps of [Ca(2+)](ER) (70 nM to 300 µM) enhanced channel activity. Thus, [Ca(2+)](ER) effects on channel activity exhibited a biphasic dependence on [Ca(2+)](i). In addition, the effect of high [Ca(2+)](ER) was attenuated when a voltage was applied to oppose Ca(2+) flux through the channel. These observations can be accounted for by Ca(2+) flux driven through the open InsP(3)R channel by [Ca(2+)](ER), raising local [Ca(2+)](i) around the channel to regulate its activity through its cytoplasmic regulatory Ca(2+)-binding sites. Importantly, [Ca(2+)](ER) regulation of InsP(3)R channel activity depended on cytoplasmic Ca(2+)-buffering conditions: it was more pronounced when [Ca(2+)](i) was weakly buffered but completely abolished in strong Ca(2+)-buffering conditions. With strong cytoplasmic buffering and Ca(2+) flux sufficiently reduced by applied voltage, both activation and inhibition of InsP(3)R channel gating by physiological levels of [Ca(2+)](ER) were completely abolished. Collectively, these results rule out Ca(2+) regulation of channel activity by direct binding to the luminal aspect of the channel.

International audience; 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.

signaling Puff termination a b s t r a c t The spatiotemporal dynamics of elementary Ca 2+ release events, such as “blips” and “puffs” shapes the hierarchal Ca2+ signaling in many cell types. Despite being the building blocks of Ca2+ patterning, the mechanism responsible for the observed properties of puffs, especially their termination is incompletely understood. In this paper, we employ a data-driven approach to gain insights into the complex dynamics of blips and puffs. We use a model of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) derived directly from single channel patch clamp data taken at 10 M concentration of IP3 to simulate calcium puffs. We first reproduce recent observations regarding puffs and blips and then investigate the mechanism of puff termination. Our model suggests that during a puff, IP3R s proceed around a loop through kinetic states from “rest” to “open” to “inhibited” and back to “rest”. A puff terminates because of self-inhibition. Based on our simulations, we rule out the endoplasmic reticulum (ER) Ca 2+ depletion as a possible cause for puff termination. The data-driven approach also enables us to estimate the current through a single IP3R and the peak Ca 2+ concentration near the channel pore.

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.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel is crucial for the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the complicated ligand regulation of this ubiquitous channel, we constructed a simple quantitative continuous-time Markov-chain model from the data. Our model accounts for most experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel activity established six main ligand-bound channel complexes, where a complex consists of one or more states with the same ligand stoichiometry and open or closed conformation. Channel gating in three distinct modes added one complex and indicated that three complexes gate in multiple modes. This also restricted the connectivity between channel complexes. Finally, latencies of channel responses to abrupt ligand concentration changes defined a model with specific network topology between 9 closed and 3 open states. The model with 28 parameters can closely reproduce the equilibrium gating statistics for all three gating modes over a broad range of ligand concentrations. It also captures the major features of channel response latency distributions. The model can generate falsifiable predictions of IP3R channel gating behaviors and provide insights to both guide future experiment development and improve IP3R channel gating analysis. Maximum likelihood estimates of the model parameters and of the parameters in the De Young–Keizer model yield strong statistical evidence in favor of our model. Our method is simple and easily applicable to the dynamics of other ion channels and molecules.

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 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 Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death., link_to_OA_fulltext

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) 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 Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R 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 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.

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.

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.

Ca2+ liberation through inositol 1,4,5-trisphosphate receptor (IP3R) channels generates complex patterns of spatiotemporal cellular Ca2+ signals owing to the biphasic modulation of channel gating by Ca2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca2+ signals (“puffs”) arising from clusters of IP3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP3R gating kinetics. To bridge these two levels of experimental data, we developed an IP3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP3R channels. The channel model consists of four identical, independent subunits, each of which has an IP3-binding site together with one activating and one inactivating Ca2+-binding site. The channel opens when at least three subunits undergo a conformational change to an “active” state after binding IP3 and Ca2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP3] and [Ca2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP3]. Assuming that stochastic opening of a single IP3R at basal cytosolic [Ca2+] and any given [IP3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP3 was obtained when the cluster contained a total of 40–70 IP3Rs.

The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP 3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP 3, but by other ligands as well, in particular cytoplasmic Ca 2+. Over the last decade, detailed quantitative studies of InsP 3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R. Copyright © 2007 the American Physiological Society., link_to_subscribed_fulltext

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.

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.

The ubiquitous, mostly endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel modulates cytoplasmic free [Ca2+] ([Ca2+]i) to generate complex Ca2+ signals by releasing Ca2+ from the ER lumen store. Whereas cytoplasmic InsP3 activation and biphasic [Ca2+]i regulation of InsP3R channel have been well studied, the effects of ER luminal free [Ca2+] ([Ca2+]ER) on InsP3R channel activity remains poorly understood and controversial. Using single-channel nuclear patch-clamp electrophysiology, we characterized a feed-through effect of [Ca2+]ER on InsP3R channel, whereby Ca2+ ion driven through an open InsP3R channel by the [Ca2+] gradient across the ER membrane can sufficiently raise the local [Ca2+]i around the channel to alter the its activity through its cytoplasmic activating and inhibitory Ca2+-binding sites. Furthermore, even in strong [Ca2+]i-buffering condition where InsP3R channel activity is not affected by Ca2+ flux across the channel, we still observed that [Ca2+]ER exerts a novel flux-independent inhibitory effect. This effect is profound, reducing InsP3R channel open probability in both saturating and sub-saturating [InsP3] by 80-90% in physiological [Ca2+]ER (∼300 μM) compared to non-physiologically low [Ca2+]ER (70 nM). The effect is observed in on-nucleus patch-clamp experiments using nuclei isolated from multiple cell types with different expression levels of various InsP3R isoforms. This effect was irreversibly abrogated when the nuclear membrane patch was excised into the luminal-side-out configuration, and the luminal side of the isolated membrane and channel was perfused with low (70 nM) [Ca2+] solution. Our observations suggest that the [Ca2+]ER inhibitory effect is likely mediated by some ER luminal peripheral accessory protein(s), which is lost during the perfusion.

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po (∼0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2–4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].

The most common form of Ca 2+ signaling by Gq-coupled receptors entails activation of PLCβ2 by Gαq to generate IP 3 and evoke Ca 2+ release from the ER [1]. Another form of Ca 2+ signaling by G protein-coupled receptors involves activation of Gi to release Gβγ, which activates PLCβ1 [2]. Whether Gβγ has additional roles in Ca 2+ signaling is unknown. Introduction of Gβγ into cells activated Ca 2+ release from the IP 3 Ca 2+ pool and Ca 2 oscillations [3, 4]. This can be due to activation of PLCβ1 or direct activation of the IP 3 R by Gβγ. We report here that Gβγ potently activates the IP 3 receptor. Thus, Gβγ-triggered [Ca 2+ ] i oscillations are not affected by inhibition of PLCβ. Coimmunoprecipitation and competition experiments with Gβγ scavengers suggest binding of Gβγ to IP 3 receptors. Furthermore, Gβγ inhibited IP 3 binding to IP 3 receptors. Notably, Gβγ activated single IP 3 R channels in native ER as effectively as IP 3 . The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca 2+ signals evoked by Gi- and Gq-coupled receptors to Gβγ scavenging and PLCβ inhibition. We propose that gating of IP 3 R by Gβγ is a new mode of Ca 2+ signaling with particular significance for Gi-coupled receptors.

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 Xenopus oocytes 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 [(14)C]methylamine (MA), had an apparent EC(50) 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 (IC(50) = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H(+)-coupled counter-transport mechanism.

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.

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP]i), here we studied the effects of [ATP]i on the dependence of r-InsP3R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i). As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 μM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is an endoplasmic reticulum–localized Ca2+-release channel that controls complex cytoplasmic Ca2+ signaling in many cell types. At least three InsP3Rs 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 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 InsP3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 μM under saturating (10 μM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP3 concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of ∼4. InsP3 activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3–induced Ca2+ release and low gain Ca2+–induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

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 wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase- and ATP-regulated chloride channel, the activity of which determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Here we describe a mechanism that regulates CFTR channel activity, which is mediated by PDZ domains, a family of conserved protein-interaction modules. The Na + /H + exchanger regulatory factor (NHERF) binds to the cytoplasmic tail of CFTR through either of its two PDZ (PDZ1 and PDZ2) domains. A recombinant fragment of NHERF (PDZ1–2) containing the two PDZ domains increases the open probability ( P o ) of single CFTR channels in excised membrane patches from a lung submucosal gland cell line. Both PDZ domains are required for this functional effect, because peptides containing mutations in either domain are unable to increase channel P o . The concentration dependence of the regulation by the bivalent PDZ1–2 domain is biphasic, i.e., activating at lower concentrations and inhibiting at higher concentrations. Furthermore, either PDZ domain alone or together is without effect on P o , but either domain can competitively inhibit the PDZ1–2-mediated stimulation of CFTR. Our results support a molecular model in which bivalent NHERF PDZ domains regulate channel gating by crosslinking the C-terminal tails in a single dimeric CFTR channel, and the magnitude of this regulation is coupled to the stoichiometry of these interactions.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](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 Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.

A Green's function approach is developed from first principles to evaluate the power spectral density of conductance fluctuations caused by ion concentration fluctuations via diffusion in an electrolyte system. This is applied to simple geometric models of transmembrane ion channels to obtain an estimate of the magnitude of ion concentration fluctuation noise in the channel current. Pure polypeptide alamethicin forms stable ion channels with multiple conductance states in artificial phospholipid bilayers isolated onto tips of micropipettes with gigaohm seals. In the single-channel current recorded by voltage-clamp techniques, excess noise was found after the background instrumental noise and the intrinsic Johnson and shot noises were removed. The noise que to ion concentration fluctuations via diffusion was isolated by the dependence of the excess current noise on buffer ion concentration. The magnitude of the concentration fluctuation noise derived from experimental data lies within limits estimated using our simple geometric channel models. Variation of the noise magnitude for alamethicin channels in various conductance states agrees with theoretical prediction.

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic [IP3] = 10 microM, free [Ca2+] = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and approximately 300 pS at 60 mV. The channel was frequently observed with high open probability (mean P(o) = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants tau5 ms and approximately 20 ms; and at least three closed states with tau approximately 1 ms, approximately 10 ms, and1 s. Higher cytoplasmic potential increased the relative frequency and tau of the longest closed state. A novel "flicker" kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (tau approximately 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (approximately 10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage-reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (1 microm) as well as macroscopic (approximately 10 microm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.

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.

Familial Alzheimer's disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutations have several cellular consequences, including exaggerated intracellular Ca2+ ([Ca2+]i) signaling - enhanced sensitivity to agonists of ER Ca2+ release and increased magnitudes of [Ca2+]i signals. Single channel recordings suggest exaggerated [Ca2+]i signaling is due to a gain-of-function interaction between FAD mutant PS and the inositol 1,4,5-trisphosphate receptor (InsP3R). However, the in vivo significance of the PS-InsP3R interaction to exaggerated [Ca2+]i signaling and the contribution of exaggerated [Ca2+]i signaling to the development of AD are unknown. If exaggerated [Ca2+]i signaling is InsP3R-dependent, we hypothesize that decreasing InsP3R protein expression will normalize [Ca2+]i signaling. Therefore, we crossed the Opisthotonus (Opt) mouse, deficient in InsP3R1 protein, to the PS1M146V knock-in (PS1KIN) AD mouse model to investigate the contribution of InsP3R-1-mediated [Ca2+]i release to exaggerated [Ca2+]i signaling, and the role of exaggerated [Ca2+]i signaling to the AD-like phenotypes of this mouse model. We observed that the Opt allele normalizes exaggerated [Ca2+]i release in vitro and ex vivo. Interestingly, in young (5wk) and mature (3 mth) PS1KIN mice we observed that the Opt allele normalized aberrantly activated hippocampal biochemical pathways and the enhancement of CA3-CA1 early long-term potentiation present in these mice - phenotypes that mirror the early hippocampal hyperactivity in patients that later develop AD. These findings indicate that FAD mutant PS-associated exaggerated [Ca2+]i signaling is InsP3R1-dependent and a early event in Alzheimer's disease.

Nuclear patch-clamp experiments can be performed with intact nuclei or with nuclei from which the outer nuclear membrane has been removed. This protocol presents procedures for harvesting different types of cultured cells, isolating nuclei, and exposing the inner nuclear membrane by agitating in the presence of sodium citrate. Particulars about obtaining and maintaining the cells of interest in culture are not described here. However, care should be taken not to allow the cells to grow beyond a density of 2–3 × 106 cells/mL because this may decrease both the cell viability and the success rate of detecting active inositol 1,4,5-trisphosphate receptor (InsP3R) channels in nuclear patches.

The modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca2+-release channels localized to the endoplasmic/sarcoplasmic reticulum—inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels—play a central role in [Ca2+]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca2+-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca2+ release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR.

Conductance noise measurement of the open states of alamethicin transmembrane channels reveals excess noise attributable to cooperative low-frequency molecular dynamics that can generate fluctuations approximately 1 A rms in the effective channel pore radius. Single-channel currents through both persistent and nonpersistent channels with multiple conductance states formed by purified polypeptide alamethicin in artificial phospholipid bilayers isolated onto micropipettes with gigaohm seals were recorded using a voltage-clamp technique with low background noise (rms noise < 3 pA up to 20 kHz). Current noise power spectra between 100 Hz and 20 kHz of each open channel state showed little frequency dependence. Noise from undetected conductance state transitions was insignificant. Johnson and shot noises were evaluated. Current noise caused by electrolyte concentration fluctuation via diffusion was isolated by its dependence on buffer concentration. After removing these contributions, significant current noise remains in all persistent channel states and increases in higher conductance states. In nonpersistent channels, remaining noise occurs primarily in the lowest two states. These fluctuations of channel conductance are attributed to thermal oscillations of the channel molecular conformation and are modeled as a Langevin translational oscillation of alamethicin molecules moving radially from the channel pore, damped mostly by lipid bilayer viscosity.

The ubiquitous, mostly endoplasmic reticulum (ER)-localized InsP3R channel modulates cytoplasmic free [Ca2+] ([Ca2+]i) to generate complex Ca2+ signals by releasing Ca2+ stored in the ER lumen. Whereas cytoplasmic InsP3 activation and biphasic [Ca2+]i regulation of the channel have been well studied, its regulation by ER luminal free [Ca2+] ([Ca2+]ER) is poorly understood and controversial. Here, we used excised luminal-side-out nuclear patch clamping with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R (InsP3R-3) channel activity. In optimal 2 μM [Ca2+]i (buffered by 0.5 mM diBrBAPTA) and sub-saturating 3 μM InsP3, jumps of [Ca2+]ER from 70 nM (no Ca2+ flux through channel) to 300 μM (substantial Ca2+ flux) reduced channel open probability (Po) significantly. This inhibition was abrogated when saturating 10 μM InsP3 was used, but was restored when [Ca2+]ER was raised to 2 mM. This [Ca2+]ER effect exhibited a biphasic dependence on [Ca2+]i because jumps of [Ca2+]ER (70 nM to 300 μM) in 3 μM InsP3 increased Po in sub-optimal 70 nM Ca2+i. These effects of [Ca2+]ER were attenuated when an electrical potential was applied to oppose Ca2+ flux through the channel. Importantly, the effects of [Ca2+]ER on channel activity depended on cytoplasmic Ca2+ buffering conditions: it was stronger when [Ca2+]i was weakly buffered by 0.1 mM HEDTA (a slow Ca2+ chelator) but was completely abolished with 5 mM diBrBAPTA buffering. These observations are completely accounted for by Ca2+ flux driven by [Ca2+]ER through the open InsP3R channel raising local [Ca2+]i around the channel to regulate its Po through its cytoplasmic activating and inhibitory Ca2+-binding sites. These results provide no evidence for a role of [Ca2+]ER in regulating InsP3R-3 activity by effects mediated directly on the luminal aspect of the channel.

The ubiquitous inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel, localized primarily in the ER membrane, releases Ca2+ into the cytoplasm upon binding IP3, generating and modulating intracellular Ca2+ signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability (Po) of the active channels, the size of the unitary Ca2+ current (iCa) passing through an open IP3R 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 IP3R channels in physiological ionic conditions has not been directly measured. Here we report the first measurement of iCa through an IP3R 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 IP3R 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 IP3R 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 IP3-liganded IP3R channels, and they confirm that Ca2+ released by an open IP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.

The versatility of Ca2+ signals derives from their spatio-temporal organization1,2. For Ca2+ signals initiated by inositol trisphosphate (IP3) this requires local interactions between IP3 receptors (IP3R)3,4 mediated by their rapid stimulation and slower inhibition4 by cytosolic Ca2+. This allows hierarchical recruitment of Ca2+ release events as the IP3 concentration increases5. Single IP3R respond first, then clustered IP3R open together giving a local Ca2+ puff, and as puffs become more frequent they ignite regenerative Ca2+ waves1,5-9. We demonstrate, using nuclear patch-clamp recording10, that IP3R are initially randomly distributed with an estimated separation of ~1 μm. Low concentrations of IP3 cause IP3R to aggregate rapidly and reversibly into small clusters of ~4 closely associated IP3R. At resting cytosolic [Ca2+], clustered IP3R open independently, but with lower open probability (Po), shorter open time, and lesser IP3 sensitivity than lone IP3R. Increasing cytosolic [Ca2+] reverses the inhibition caused by clustering, IP3R gating becomes coupled, and the duration of multiple openings is prolonged. Clustering both exposes IP3R to local Ca2+ rises and increases the effects of Ca2+. Dynamic regulation of clustering by IP3 tunes IP3R sensitivity to IP3 and Ca2+, facilitating hierarchical recruitment of the elementary events that underlie all IP3-evoked Ca2+ signals3,5.

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 bymechanisms 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 they were more likely than IP 3R 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. Copyright 2008 by the American Association for the Advancement of Science; all rights reserved., link_to_subscribed_fulltext

Publisher Summary The activity of the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ release channel is intricately regulated by a multitude of ligands. The most important ligands regulating IP3R channel activity are IP3 and Ca2+. Generally, cytoplasmic Ca2+ regulates steady-state IP3R channel gating with a biphasic concentration dependence—Ca2+ at low concentrations activates the channel and increases its open probability, whereas at higher concentrations, Ca2+ inhibits the channel. Ca2+ modulates channel activity by binding to several apparently distinct sites that regulate Ca2+activation, IP3-dependent Ca2+ inhibition, IP3-independent Ca2+ inhibition, channel inactivation, and channel recruitment. A separate Ca2+-binding site appears to regulate the functionality of the IP3-independent Ca2+ inhibition site. Quantitative and qualitative molecular models can account for much of the steady-state and kinetic behaviors of IP3R channel gating regulation by cytoplasmic Ca2+. The identification of modal gating has revealed that Ca2+modulates IP3R channel activity primary by regulating its propensity to gate in particular modes. Ca2+ regulation of the channel is impinged on by other channel regulators, including IP3 and ATP, as allosteric modulators.

The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel is engaged by thousands of plasma membrane receptors to generate Ca(2+) signals in all cells. Understanding how complex Ca(2+) signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP(3) and Ca(2+), which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP(3)R 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 Ca(2+) regulation and unexpected ligand cooperativity. The kinetics of Ca(2+)-mediated channel inhibition showed the single-channel bases for fundamental Ca(2+) release events and Ca(2+) release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca(2+) signals.

The inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) plays a critical role in generation of complex Ca 2+ signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP 3R channels were consistently detected with regulation by cytoplasmic InsP 3 and free Ca 2+ concentrations ([Ca 2+] i) very similar to that observed for vertebrate InsP 3R. Long channel activity durations of the Sf9-InsP 3R have now enabled identification of a novel aspect of InsP 3R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP 3R 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 [Ca 2+] i and [InsP 3] does not substantially alter channel P o within a mode. Instead, [Ca 2+] i and [InsP 3] 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 InsP 3R channel activity, with implications for the kinetics of Ca 2+ release events in cells. © The Rockefeller University Press., published_or_final_version

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 (N(A)) to be accurately quantified. InsP3-activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca2+]i and suboptimal [InsP3]. Unexpectedly, N(A) 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.

In many cell-signaling pathways, information is transmitted by the diffusion of messenger molecules. Diffusion coefficients characterize the messenger's spatial range and the characteristic times of signal propagation. Inside cells, particles usually diffuse in the presence of immobile binding sites (or traps). It is well known that binding to traps results in an effective diffusion coefficient that is smaller than the free coefficient in media free of traps. To measure effective diffusion coefficients in cells, "tagged" particles are often used. Radioactive calcium was used in a giant squid axon and in cytosolic extracts of Xenopus laevis oocytes. Fluorescence recovery after photobleaching yields diffusion coefficients from observations of the distribution of fluorescently labeled proteins. In the absence of traps, free diffusion coefficients give both the rate at which single-particle mean square displacements increase and the rate at which information in the form of inhomogeneities in particle concentration spread out with time. We show here that, in the presence of traps, information diffuses faster than single particles. Thus, messages diffuse faster than messengers. Tagged-particle experiments give the single-particle diffusion coefficients and, thus, can underestimate the rate of diffusive signal propagation. © 2006 by The National Academy of Sciences of the USA. Fil: Pando, Bernardo. Massachusetts Institute of Technology; Estados Unidos Fil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Mak, Don-On Daniel. University of Pennsylvania; Estados Unidos Fil: Pearson, John E.. Los Alamos National Laboratory