scholarly journals Regulation of the Type 1 and Type 2 Inositol 1,4,5-Trisphosphate Receptor Single Channel Open Probability by ATP in On-Nucleus Patch Clamp Recordings

2011 ◽  
Vol 100 (3) ◽  
pp. 250a
Author(s):  
Larry E. Wagner ◽  
David I. Yule
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Open Probability ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

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 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.

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.

scholarly journals Cardiac Type 2 Inositol 1,4,5-Trisphosphate Receptor

2005 ◽  
Vol 280 (16) ◽  
pp. 15912-15920 ◽  
Author(s):  
Dan J. Bare ◽  
Claudia S. Kettlun ◽  
Mei Liang ◽  
Donald M. Bers ◽  
Gregory A. Mignery
Keyword(s):  
Lipid Bilayers ◽  
Calcium Release ◽  
Ventricular Myocytes ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Release Channel ◽  
Amino Terminal ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor

The type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP3R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP3R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP3R2 associates with Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ), the major isoform expressed in cardiac myocytes. Recombinant InsP3R2 and CaMKIIδBalso co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP3R2 were sufficient for interaction with CaMKIIδBand associated upon mixing following separate expression. CaMKII can also phosphorylate InsP3R2, as demonstrated by32P labeling. Incorporation of CaMKII-treated InsP3R2 into planar lipid bilayers revealed that InsP3-mediated channel open probability is significantly reduced (∼11 times) by phosphorylation via CaMKII. We concluded that the InsP3R2 and CaMKIIδ likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP3R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP3R2 function.

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 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 Rac1-mediated NADPH oxidase release of O2− regulates epithelial sodium channel activity in the alveolar epithelium

2010 ◽  
Vol 298 (4) ◽  
pp. L509-L520 ◽  
Author(s):  
Yoshizumi Takemura ◽  
Preston Goodson ◽  
Hui Fang Bao ◽  
Lucky Jain ◽  
My N. Helms
Keyword(s):  
Nadph Oxidase ◽  
Sodium Channel ◽  
Single Channel ◽  
Epithelial Sodium Channel ◽  
Alveolar Epithelial Cells ◽  
Apical Surface ◽  
Alveolar Cells ◽  
Open Probability

We examine whether alveolar cells can control release of O2− through regulated NADPH oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis. Using FACS to purify alveolar epithelial cells, we show that type 1 cells robustly express each of the critical NOX components that catalyze the production of O2− (NOX2 or gp91 phox, p22 phox, p67 phox, p47 phox, and p40 phox subunits) as well as Rac1 at substantially higher levels than type 2 cells. Immunohistochemical labeling of lung tissue shows that Rac1 expression is cytoplasmic and resides near the apical surface of type 1 cells, whereas NOX2 coimmunoprecipitates with epithelial sodium channel (ENaC). Since Rac1 is a known regulator of NOX2, and hence O2− release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 μM NSC23766 inhibition of Rac1 decreased O2− output in lung cells and significantly decreased ENaC activity from 0.87 ± 0.16 to 0.52 ± 0.16 [mean number of channels ( N) and single-channel open probability ( Po) ( NPo) ± SE, n = 6; P < 0.05] in type 2 cells. NSC23766 (10 μM) decreased ENaC NPo from 1.16 ± 0.27 to 0.38 ± 0.10 ( n = 6 in type 1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1 and O2− release) increased ENaC NPo values in both type 1 and 2 cells. NPo values increased from 0.48 ± 0.21 to 0.91 ± 0.28 in type 2 cells ( P < 0.05; n = 10). In type 1 cells, ENaC activity also significantly increased from 0.40 ± 0.15 to 0.60 ± 0.23 following EGF treatment ( n = 7). Sequestering O2− using 2,2,6,6-tetramethylpiperidine- N-oxyl (TEMPO) compound prevented EGF activation of ENaC in both type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2 activity is an important component in O2− regulation of ENaC.

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.

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