Dataset Information

Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

ABSTRACT: Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP₃Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP₃R1, focusing on the ∼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914-1926) that is predicted to contain two fused, disjointed transient helical elements. Experiments with IP₃R1 mutants, combined with computational analysis, indicated that small deletions in this central region block Bok binding due to perturbation of the helical elements. Studies in vitro with purified Bok and IP₃R1-derived peptides revealed high affinity binding to amino acids 1898-1940 of IP₃R1 (K_d ∼65 nM) and that binding affinity is also dependent upon both of the high disorder flanking regions. The strength of the Bok-IP₃R1 interaction was demonstrated by the ability of IP₃R1 or Bok to recruit transmembrane domain-free Bok or IP₃R1 mutants, respectively, to membranes in intact cells, and that these two mutants can bind in the cytosol independently of membrane association. Overall, we show that Bok binding to IP₃R1 occurs within a largely disordered loop between α helices 72 and 73 and that high affinity binding is mediated by multivalent interactions.

SUBMITTER: Szczesniak LM

PROVIDER: S-EPMC8052301 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

Szczesniak Laura M LM Bonzerato Caden G CG Schulman Jacqualyn J JJ Bah Alaji A Wojcikiewicz Richard J H RJH

Biochemical and biophysical research communications 20210324

Bcl-2-related ovarian killer (Bok) binds tightly to inositol 1,4,5-trisphosphate receptors (IP<sub>3</sub>Rs). To better understand this interaction, we sought to elucidate the Bok binding determinants in IP<sub>3</sub>R1, focusing on the ∼75 amino acid loop (residues 1882-1957) between α helices 72 and 73. Bioinformatic analysis revealed that the majority of this loop is intrinsically disordered, with two flanking regions of high disorder next to a low disorder central region (∼residues 1914-19 ...[more]

PMID: 33773141

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Project description:The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 μmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 μmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 μM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 μmol/L). In saponin permeabilized AMs InsP3 (5 μmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100μm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100μm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Dataset Information

Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

Publications

Bok binds to a largely disordered loop in the coupling domain of type 1 inositol 1,4,5-trisphosphate receptor.

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