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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.

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.

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.

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.

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.

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 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.

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.

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.

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

Familial Alzheimer’s disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aβ) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca2+ signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca2+ signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca2+ and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R 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, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca2+ concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca2+ and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca2+ signals, which can account for the more frequent global Ca2+ signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca2+ signals in cells expressing FAD-causing mutant PS., Author Summary Aberrant Ca2+ signaling caused by IP3R gating dysregulation is implicated in many neurodegenerative diseases such as Alzheimer’s, Huntington’s, Spinocerebellar ataxias, and endoplasmic reticulum stress-induced brain damage. Thus understanding IP3R dysfunction is important for the etiology of these diseases. It was previously shown that FAD-causing mutant PS interacts with the IP3R, leading to its gain-of-function enhancement in optimal Ca2+ and sub-saturating IP3 concentrations. Here, we use data-driven modeling to provide deeper insights into the upregulation of IP3R gating in a wide range of ligand concentrations and quantify the sensitivity of the channel to its ligands in the presence of mutant PS. Our simulations demonstrate that these changes can alter the statistics of local Ca2+ events and we speculate that they lead to Ca2+ signaling dysregulations at the whole cell level observed in FAD cells. These models will provide the foundation for future data-driven computational framework for local and global Ca2+ signals that will be used to judiciously isolate the primary pathways causing Ca2+ dysregulation in FAD from those that are downstream, and to study the effects of upregulation of IP3R activity on cell function.

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

The erythrocyte Rh-associated glycoprotein (RhAG) was recently found to mediate transport of ammonia/ammonium when expressed in Xenopus laevis oocytes and yeast Saccharomyces cerevisiae. Nonerythroid homologs, RhBG and RhCG, are expressed in the mammalian kidney connecting segment and the collecting duct, major sites of urinary ammonia secretion. This study characterizes the transport properties of murine RhBG and RhCG by ammonium analog [14C]methylamine (MA) uptake and two-electrode voltage clamping of X. laevis oocytes. Both RhBG and RhCG mediated transport of ammonia, but differed in affinity for substrate ( Km= 2.5 and 10 mM, respectively). The rates of RhBG- and RhCG-mediated transport were sensitive to the concentration of the protonated MA species and were stimulated by extracellular alkalosis and inhibited by acidosis, suggesting a role for H+in the transport process. Whereas expression of RhBG or RhCG caused a small increase in plasma membrane conductance, [14C]MA uptake was not affected by depolarization of oocytes with 100 mM extracellular K+or by clamping the membrane potential between 0 and −100 mV, indicating that RhBG- and RhCG-mediated transport was independent of the membrane potential. These results strongly suggest that RhBG and RhCG transport ammonia by an electroneutral process that involves NH4+/H+exchange resulting in net NH3translocation. The polarized localization of RhBG and RhCG in kidney tubules and the different substrate affinities may enable these proteins to participate in transepithelial ammonia secretion and to therefore play an important role in whole animal acid-base regulation.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.

Single-channel activities of homotetrameric recombinant rat type 1 InsP3R channels expressed in mutant DT40-3KO cells with no endogenous InsP3R expression were studied in nuclear patch clamp experiments. In on-nucleus experiments with steady ligand conditions, the channel exhibits modal gating with at least two modes: a high-Po mode with Po = 0.4-0.6 and mean open duration (to) ∼ 3 ms; and a low-Po mode with Po < 0.05 but to still ∼ 3 ms. Cytoplasmic free Ca2+ concentration ([Ca2+]i) regulates channel Po biphasically by changing the likelihood of the channel being in the high-Po mode and the mean closed duration (tc) of the low-Po mode. Even under optimal ligand conditions ([Ca2+]i = 2 µM and cytoplasmic InsP3 concentration [InsP3] = 10 µM), the maximum mean Po observed was only ∼ 0.1, with the channel being in the low-Po mode the majority of time. InsP3 (33 nM to 10 µM) activates the channel by reducing its sensitivity to Ca2+ inhibition. In experiments with cytoplasmic-side-out nuclear membrane patches, when initial [Ca2+]i was low (55 nM) and [InsP3] was rapidly dropped from 10 µM to 0 using perfusion solution exchanges, the InsP3R channels had a high propensity to transiently enter a more active state before they became inactivated by the removal of InsP3. This behavior was more prominent, with the channel Po transiently increasing to ∼ 0.6, when [Ca2+]i was increased to optimal level (2.1 µM) instead of remaining constant at 55 nM. No transient increase in Po was observed in similar [InsP3] drops with [Ca2+]i kept constant at 2.1 µM. This suggests that sub-saturating [InsP3] can induce type 1 InsP3R channels to enter the high-Po mode with higher probability than saturating [InsP3].

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R 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 InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R 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 Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.

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 contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 Ins P 3 R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, Ins P 3 R-mediated Ca 2+ signaling in cells is possible even in the absence of Ins P 3 generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca 2+ concentration by Ins P 3 -independent pathways and for localizing Ins P 3 -mediated Ca 2+ signals to individual synapses.

Inositol 1,4,5-tris-phosphate (IP 3 ) binding to its receptors (IP 3 R) in the endoplasmic reticulum (ER) activates Ca 2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca 2+ concentration signals including temporal oscillations and propagating waves. IP 3 -mediated Ca 2+ release is also controlled by cytoplasmic Ca 2+ concentration with both positive and negative feedback. Single-channel properties of the IP 3 R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP 3 R on cytoplasmic Ca 2+ and IP 3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca 2+ concentration ([Ca 2+ ] i ) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP 3 , even as [Ca 2+ ] i was raised to high concentrations, displaying two distinct types of functional Ca 2+ binding sites: activating sites with half-maximal activating [Ca 2+ ] i ( K act ) of 210 nM and Hill coefficient ( H act ) ≈2; and inhibitory sites with half-maximal inhibitory [Ca 2+ ] i ( K inh ) of 54 μM and Hill coefficient ( H inh ) ≈4. Lowering IP 3 concentration was without effect on Ca 2+ activation parameters or H inh , but decreased K inh with a functional half-maximal activating IP 3 concentration ( K IP 3 ) of 50 nM and Hill coefficient ( H IP 3 ) of 4 for IP 3 . These results demonstrate that Ca 2+ is a true receptor agonist, whereas the sole function of IP 3 is to relieve Ca 2+ inhibition of IP 3 R. Allosteric tuning of Ca 2+ inhibition by IP 3 enables the individual IP 3 R Ca 2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca 2+ concentration signaling and quantal Ca 2+ release.

The effects of Mg2+and Ba2+on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopusoocytes. In 140 mM K+the IP3R channel kinetics and presence of conductance substates were similar over a range (0–9.5 mM) of free Mg2+. In 0 mM Mg2+the channel current-voltage ( I-V) relation was linear with conductance of ∼320 pS. Conductance varied slowly and continuously over a wide range (SD ≈ 60 pS) and sometimes fluctuated during single openings. The presence of Mg2+on either or both sides of the channel reduced the current (blocking constant ∼0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ∼120 pS near 0 mV and ∼360 pS at ±70 mV in symmetrical 2.5 mM Mg2+). Ba2+exhibited similar effects on channel conductance. Mg2+and Ba2+permeated the channel with a ratio of permeability of Ba2+to Mg2+to K+of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel plays a central role in the generation and modulation of intracellular Ca2+ signals in animal cells. To gain insight into the crucial intricate ligand regulation of this ubiquitous channel, we constructed the simplest quantitative continuous-time Markov-chain model using a transparent iterative approach, which can be adopted to Markov chain models in general. Our model accounts for all experimentally observed gating behaviors of single native IP3R channels from insect Sf9 cells. Ligand (Ca2+ and IP3) dependencies of channel open probability (PO) 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 additional complex and indicated that three of the complexes can gate in two different modes. This also restricted the connectivity between channel complexes. Finally, channel responses to abrupt ligand concentration changes defined a model with 9 closed states and 3 open states, and its network topology. The model with 24 parameters can closely reproduce the equilibrium PO and channel gating statistics for all three gating modes for 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 not yet explored, and provide insights to both guide future experiment development and improve IP3R channel gating analysis.

Molecular structures of transmembrane channels formed by alamethicin polypeptide aggregates were analyzed by measuring open-channel conductances and state-transition kinetics using voltage-clamp technique with artificial phospholipid bilayers isolated onto micropipettes by a novel solvent-free tip-dip method. Two distinct classes of alamethicin channels, each with a unique set of conductance states and kinetic properties, were identified. Alamethicin Rf50 at low temperatures forms mostly nonpersistent channels with lifetimes of < 1 min. Long-lasting persistent channels are formed by alamethicin Rf30 at all temperatures and by alamethicin Rf50 at room temperature. In the "modified barrel-stave" model for persistent channels based on the crystalline alamethicin secondary structure, the aqueous pore of the channel surrounded by parallel alamethicin monomers has a constriction generated by amino acid side chains protruding from the alamethicin helices into the pore. The model explains quantitatively the nonohmic channel conductance at high applied voltages and the conductance values and ion selectivities of various persistent channel states. The kinetic properties of nonpersistent channels are explained qualitatively by the "reversed-molecule" model in which nonpersistent channels differ from persistent channels by having one of the channel-forming alamethicin monomers oriented antiparallel to the others.

Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-dependent channel activity in only 1.5% of patches. With K(+) (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.