scholarly journals General Anesthetic Isoflurane Modulates Inositol 1,4,5-Trisphosphate Receptor Calcium Channel Opening

2014 ◽  
Vol 121 (3) ◽  
pp. 528-537 ◽  
Author(s):  
J. Donald Joseph ◽  
Yi Peng ◽  
Don-On Daniel Mak ◽  
King-Ho Cheung ◽  
Horia Vais ◽  
...  
Keyword(s):  
Single Channel ◽  
General Anesthetics ◽  
General Anesthetic ◽  
Minimal Alveolar Concentration ◽  
Open Probability ◽  
Recombinant Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

Abstract 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.

scholarly journals Location of the Permeation Pathway in the Recombinant Type 1 Inositol 1,4,5-Trisphosphate Receptor

1999 ◽  
Vol 114 (2) ◽  
pp. 243-250 ◽  
Author(s):  
Josefina Ramos-Franco ◽  
Daniel Galvan ◽  
Gregory A. Mignery ◽  
Michael Fill
Keyword(s):  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Single Channel ◽  
Site Directed Mutagenesis ◽  
Recombinant Type ◽  
Mutation Strategy ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Permeation Properties

The inositol 1,4,5-trisphosphate receptor (InsP3R) forms ligand-regulated intracellular Ca2+ release channels in the endoplasmic reticulum of all mammalian cells. The InsP3R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP3R pore. Mutant InsP3Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; PCa/PCs = 6.3). These mutant channels bound InsP3, but ligand occupancy did not regulate the constitutively open pore (Po > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398–2589) near the COOH terminus of the protein forms the InsP3R pore. Further, we have produced a constitutively open InsP3R pore mutant that is ideal for future site-directed mutagenesis studies of the structure–function relationships that define Ca2+ permeation through the InsP3R channel.

Halothane-induced synaptic depression at both in vivo and in vitro reconstructed synapses between identified Lymnaea neurons

1995 ◽  
Vol 74 (6) ◽  
pp. 2604-2613 ◽  
Author(s):  
G. E. Spencer ◽  
N. I. Syed ◽  
K. Lukowiak ◽  
W. Winlow
Keyword(s):  
Synaptic Transmission ◽  
Lymnaea Stagnalis ◽  
Cell Types ◽  
Inhibitory Synapses ◽  
General Anesthetics ◽  
General Anesthetic ◽  
Presynaptic Neuron ◽  
Novel Approach

1. In the present study we tested the ability of the general anesthetic, halothane, to affect synaptic transmission at in vivo and in vitro reconstructed peptidergic synapses between identified neurons of Lymnaea stagnalis. 2. An identified respiratory interneuron, visceral dorsal 4 (VD4), innervates a number of postsynaptic cells in the central ring ganglia of Lymnaea. Because VD4 has previously been shown to exhibit immunoreactivity for FMRFamide-related peptides, it was hypothesized that these peptides may be utilized by VD4 during synaptic transmission. In the intact, isolated CNS of Lymnaea, we have identified novel connections between VD4 and the pedal A (PeA) cells. We demonstrate that VD4 makes inhibitory connections with the PeA neurons, in particular PeA4, and that these synaptic responses are mimicked by exogenous application of FMRFamide. 3. The synaptic transmission between VD4 and the PeA cells in an intact, isolated CNS preparation was completely blocked in 2%, but not 1% halothanc. Interestingly, the postsynaptic responses (PeA) to exogenous FMRFamide were maintained in the presence of both 1 and 2% halothane. 4. To determine the specificity of the observed responses and to determine the precise synaptic site of anesthetic action, we reconstructed the VD4/PeA synapses in vitro. After isolation from their respective ganglia, both cell types extended processes and established neuritic contact. We demonstrated that not only did the presynaptic neuron reestablish the appropriate inhibitory synapses with the PeA neurons, but that the PeA cells also maintained their responsiveness to exogenous FMRFamide. 5. Superfusion of the in vitro synaptically connected VD4 and PeA cells with 2% halothane completely abolished the synaptic transmission between these cells. However, even higher concentrations of 4% halothane failed to block the responsiveness of the PeA neurons to exogenous FMRFamide. Moreover, both 1 and 2% halothane enhanced the duration of the postsynaptic response to exogenously applied FMRFamide. These data suggest that the halothane-induced depression of synaptic transmission most likely occurred at the presynaptic level. 6. This study provides the first direct evidence that peptidergic transmission in the nervous system may also be susceptible to the actions of general anesthetics. In addition, we utilized a novel approach of in vitro reconstructed synapses for studying the effects of general anesthetics on monosynaptic transmission in the absence of other synaptic influences.

scholarly journals Isoform-specific Regulation of the Inositol 1,4,5-Trisphosphate Receptor by O-Linked Glycosylation

2011 ◽  
Vol 286 (18) ◽  
pp. 15688-15697 ◽  
Author(s):  
Patricia Bimboese ◽  
Craig J. Gibson ◽  
Stefan Schmidt ◽  
Wanqing Xiang ◽  
Barbara E. Ehrlich
Keyword(s):  
Intracellular Calcium ◽  
Cell Line ◽  
Calcium Release ◽  
Single Channel ◽  
Open Probability ◽  
Functional Changes ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cholangiocarcinoma Cell ◽  
Specific Regulation ◽  
Trisphosphate Receptor

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP3R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813–13821). We now report the effect of O-GlcNAcylation on InsP3R-2 and InsP3R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP3R-2, showed no detectable O-GlcNAcylation of InsP3R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP3R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP3R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP3R-3 channel was opposite the effect measured with InsP3R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP3R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP3-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP3R isoform specificity suggest that this form of modification of InsP3R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.

scholarly journals ATP Regulation of Type-1 Inositol 1,4,5-Trisphosphate Receptor Activity Does Not Require Walker A-type ATP-binding Motifs

2009 ◽  
Vol 284 (24) ◽  
pp. 16156-16163 ◽  
Author(s):  
Matthew J. Betzenhauser ◽  
Larry E. Wagner ◽  
Hyung Seo Park ◽  
David I. Yule
Keyword(s):  
Single Channel ◽  
Mutational Analysis ◽  
Atp Binding ◽  
Wild Type ◽  
Open Probability ◽  
Permeabilized Cells ◽  
Atp Binding Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

scholarly journals Single-Channel Function of Recombinant Type 2 Inositol 1,4,5-Trisphosphate Receptor

2000 ◽  
Vol 79 (3) ◽  
pp. 1388-1399 ◽  
Author(s):  
Josefina Ramos-Franco ◽  
Dan Bare ◽  
Sean Caenepeel ◽  
Alma Nani ◽  
Michael Fill ◽  
...  
Keyword(s):  
Single Channel ◽  
Channel Function ◽  
Recombinant Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

scholarly journals The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

1998 ◽  
Vol 330 (1) ◽  
pp. 559-564 ◽  
Author(s):  
C. Edwin THROWER ◽  
J. A. Edward LEA ◽  
P. Alan DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

scholarly journals Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

2007 ◽  
Vol 407 (2) ◽  
pp. 303-311 ◽  
Author(s):  
Benoit Devogelaere ◽  
Monique Beullens ◽  
Eva Sammels ◽  
Rita Derua ◽  
Etienne Waelkens ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Phosphorylation Sites ◽  
Docking Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Receptor Binding Protein

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1–104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.

Enhanced Thalamic Spillover Inhibition during Non–rapid-eye-movement Sleep Triggers an Electrocortical Signature of Anesthetic Hypnosis

2016 ◽  
Vol 125 (5) ◽  
pp. 964-978 ◽  
Author(s):  
Lia Mesbah-Oskui ◽  
Richard L. Horner
Keyword(s):  
Eye Movement ◽  
Rem Sleep ◽  
Aminobutyric Acid ◽  
Rapid Eye Movement ◽  
General Anesthetics ◽  
General Anesthetic ◽  
Brain Wave ◽  
Electrocortical Activity

Abstract Background Alterations in thalamic γ-aminobutyric acid–mediated signaling are thought to underlie the increased frontal α-β frequency electrocortical activity that signals anesthetic-induced loss of consciousness with γ-aminobutyric acid receptor type A (GABAAR)–targeting general anesthetics. The general anesthetic etomidate elicits phasic extrasynaptic GABAAR activation (“spillover” inhibition) at thalamocortical neurons in vitro. We hypothesize that this action of etomidate at the thalamus is sufficient to trigger an increase in frontal α-β frequency electrocortical activity and that this effect of etomidate is fully recapitulated by enhanced thalamic spillover inhibition in vivo. Methods We recorded electrocortical activity and sleep–wake behavior in freely behaving wild-type (n = 33) and extrasynaptic δ-subunit–containing GABAAR knockout mice (n = 9) during bilateral microperfusion of the thalamus with etomidate and/or other pharmacologic agents that influence GABAAR or T-type Ca2+ channel activity. Results Microperfusion of etomidate into the thalamus elicited an increase in α-β frequency electrocortical activity that occurred only during non–rapid-eye-movement (REM) sleep (11.0 ± 11.8% and 16.0 ± 14.2% greater 8 to 12- and 12 to 30-Hz power, respectively; mean ± SD; both P < 0.031) and was not affected by blockade of thalamic T-type Ca2+ channels. Etomidate at the thalamus also increased spindle-like oscillations during non-REM sleep (4.5 ± 2.4 spindle per minute with etomidate vs. 3.2 ± 1.7 at baseline; P = 0.002). These effects of etomidate were fully recapitulated by enhanced thalamic extrasynaptic GABAAR-mediated spillover inhibition. Conclusions These findings identify how a prototypic GABAAR-targeting general anesthetic agent can elicit the characteristic brain wave pattern associated with anesthetic hypnosis when acting at the thalamus by promoting spillover inhibition and the necessity of a preexisting non-REM mode of activity in the thalamus to generate this effect.

scholarly journals Calpain-cleaved Type 1 Inositol 1,4,5-Trisphosphate Receptor (InsP3R1) Has InsP3-independent Gating and Disrupts Intracellular Ca2+ Homeostasis

2011 ◽  
Vol 286 (41) ◽  
pp. 35998-36010 ◽  
Author(s):  
Catherine M. Kopil ◽  
Horia Vais ◽  
King-Ho Cheung ◽  
Adam P. Siebert ◽  
Don-On Daniel Mak ◽  
...  
Keyword(s):  
Single Channel ◽  
Open Probability ◽  
Release Channel ◽  
Terminal Fragment ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Functional Consequences ◽  
Carboxyl Terminal ◽  
Trisphosphate Receptor

The type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) is a ubiquitous intracellular Ca2+ release channel that is vital to intracellular Ca2+ signaling. InsP3R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca2+ homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP3R1 and utilized a recombinant truncated form of the channel (capn-InsP3R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP3R1 revealed InsP3-independent gating and high open probability (Po) under optimal cytoplasmic Ca2+ concentration ([Ca2+]i) conditions. However, some [Ca2+]i regulation of the cleaved channel remained, with a lower Po in suboptimal and inhibitory [Ca2+]i. Expression of capn-InsP3R1 in N2a cells reduced the Ca2+ content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca2+ loading compared with control cells expressing full-length InsP3R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP3R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP3R1 generates a dysregulated channel that disrupts cellular Ca2+ homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP3R1 in a clinically relevant injury model, suggesting that Ca2+ leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.

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