scholarly journals Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors

1997 ◽  
Vol 322 (2) ◽  
pp. 591-596 ◽  
Author(s):  
Hiroyuki YONESHIMA ◽  
Atsushi MIYAWAKI ◽  
Takayuki MICHIKAWA ◽  
Teiichi FURUICHI ◽  
Katsuhiko MIKOSHIBA
Keyword(s):  
Binding Sites ◽  
Functional Difference ◽  
Binding Assays ◽  
Binding Properties ◽  
Ligand Affinity ◽  
Kd Values ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insect Ovary ◽  
Type 3

To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 μM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 μM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 μM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 μM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.

Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP

Cell Calcium ◽  
2000 ◽  
Vol 27 (5) ◽  
pp. 257-267 ◽  
Author(s):  
K. Maes ◽  
L. Missiaen ◽  
P. De Smet ◽  
S. Vanlingen ◽  
G. Callewaert ◽  
...  
Keyword(s):  
Receptor Type ◽  
Differential Modulation ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Type 3

scholarly journals Importance of Cry1 δ-Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens(L.)

2001 ◽  
Vol 67 (1) ◽  
pp. 323-329 ◽  
Author(s):  
Juan Luis Jurat-Fuentes ◽  
Michael J. Adang
Keyword(s):  
Binding Sites ◽  
Heliothis Virescens ◽  
Brush Border Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Cross Resistance ◽  
Binding Assays ◽  
Binding Properties ◽  
Toxin Binding ◽  
Cry Toxin

ABSTRACT We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with 125I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [K com] = 1.1 nM) for 125I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for125I-Cry1Ab binding sites, though theK com values ranged from 179 to 304 nM. Cry1Ab competed for 125I-Cry1Ac binding sites (K com = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the 125I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.

scholarly journals Regulation by Ca2+ and Inositol 1,4,5-Trisphosphate (Insp3) of Single Recombinant Type 3 Insp3 Receptor Channels

2001 ◽  
Vol 117 (5) ◽  
pp. 435-446 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Sean McBride ◽  
J. Kevin Foskett
Keyword(s):  
Mammalian Cells ◽  
Single Channel ◽  
Channel Gating ◽  
Hill Coefficient ◽  
Release Channel ◽  
Recombinant Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Type 3 ◽  
Insp3 Receptor

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.

scholarly journals Differential Modulation of Type-1, Type-2 and Type-3 Inositol (1,4,5)-Trisphosphate Receptors by ATP

2009 ◽  
Vol 96 (3) ◽  
pp. 560a
Author(s):  
Matthew J. Betzenhauser ◽  
Larry E. Wagner ◽  
Hyung Seo Park ◽  
Lyndee Knowlton ◽  
David I. Yule
Keyword(s):  
Differential Modulation ◽  
Inositol 1,4,5 Trisphosphate ◽  
Type 3

scholarly journals von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy

Blood ◽  
2013 ◽  
Vol 122 (23) ◽  
pp. 3735-3740 ◽  
Author(s):  
David Lillicrap
Keyword(s):  
Molecular Genetic ◽  
Diagnostic Algorithm ◽  
Von Willebrand Disease ◽  
Functional Assay ◽  
Binding Assays ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand disease (VWD) is the most common autosomally inherited bleeding disorder. The disease represents a range of quantitative and qualitative pathologies of the adhesive glycoprotein von Willebrand factor (VWF). The pathogenic mechanisms responsible for the type 2 qualitative variants of VWF are now well characterized, with most mutations representing missense substitutions influencing VWF multimer structure and interactions with platelet GPIbα and collagen and with factor VIII. The molecular pathology of type 3 VWD has been similarly well characterized, with an array of different mutation types producing either a null phenotype or the production of VWF that is not secreted. In contrast, the pathogenetic mechanisms responsible for type 1 VWD remain only partially resolved. In the hemostasis laboratory, the measurement of VWF:Ag and VWF:RCo are key components in the diagnostic algorithm for VWD, although the introduction of direct GPIbα-binding assays may become the functional assay of choice. Molecular genetic testing can provide additional benefit, but its utility is currently limited to type 2 and 3 VWD. The treatment of bleeding in VWD involves the use of desmopressin and plasma-derived VWF concentrates and a variety of adjunctive agents. Finally, a new recombinant VWF concentrate has just completed clinical trial evaluation and has demonstrated excellent hemostatic efficacy and safety.

Expression of inositol 1,4,5-trisphosphate receptors changes the Ca2+ signal of Xenopus oocytes

1996 ◽  
Vol 270 (4) ◽  
pp. C1255-C1261 ◽  
Author(s):  
S. DeLisle ◽  
O. Blondel ◽  
F. J. Longo ◽  
W. E. Schnabel ◽  
G. I. Bell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Expression Level ◽  
Xenopus Laevis Oocytes ◽  
Biological Functions ◽  
Propagating Waves ◽  
Inositol 1,4,5 Trisphosphate ◽  
Related Proteins ◽  
Type 3

The receptors for the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] form a family of closely related proteins that play an important role in regulating the free intracellular Ca2+ concentration. To test the hypothesis that changing the expression level of Ins(1,4,5)P3 receptors could alter the Ins(1,4,5)P3-mediated Ca2+ signal, we overexpressed Ins(1,4,5)P3 receptor type 1 (InsP3R-1) or type 3 (InsP3R-3) in Xenopus laevis oocytes. Expression of InsP3R-1 increased the velocity of the propagating waves of intracellular Ca2+ release but did not affect the Ins(1,4,5)P3-induced entry of extracellular Ca2+ across the plasma membrane. In contrast, expression of intracellular Ca2+ but markedly increased the magnitude and duration of Ca2+ influx. Immunolocalization studied revealed InsP3R-3 at the endoplasmic reticulum, with a relatively stronger signal at or near the plasma membrane. The results suggest that changing the expression level of an InsP3R can alter the Ins(1,4,5)P3-mediated Ca2+ signal and that InsP3R-1 and InsP3R-3 may have different biological functions.

Effects of Single Point Mutations within Proposed Copper Binding Sites on Specific Activity and Inter-Chain Affinity of Factor VIII.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1730-1730
Author(s):  
Hironao Wakabayashi ◽  
Qian Zhou ◽  
Keiji Nogami ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Specific Activity ◽  
Single Point ◽  
Point Mutations ◽  
High Affinity ◽  
Kd Values ◽  
Single Point Mutations

Abstract Copper ions appear important for the structural integrity and the function of factor VIII. Reconstitution studies have shown that Cu2+ increases specific activity of factor VIII as well as increases affinity between heavy chain (HC) and light chain (LC). Based on the ceruloplasmin homology, the existence of three Cu2+ binding sites has been proposed. These include a type 1 site in HC (A1 domain) coordinated by C310, H315, H267, and M320, a type 1 site in LC (A3 domain) coordinated by C2000, H1954, H2005, and M2010, and type 2 site spanning A1 and A3 domains coordinated by H99 and H1957 with possible other additional residues. We rationalized that point mutations within a putative Cu2+ coordination sites would diminish Cu2+ binding at that site, as detected by changes in factor VIII specific activity and/or inter-subunit affinity. We produced several mutant factor VIII proteins bearing point mutation of C310S, H315A, C2000S, H1954A, H99A, or H1957A using a B-domainless human factor VIII vector. Each mutant was stably expressed and purified by SP-sepharose, which bound factor VIII primarily though LC. Western blotting indicated a reduction in the relative amount of HC in C310S and H99A factor VIII forms, suggesting that inter-chain affinity was reduced by these mutations. EDTA-treated factor VIII forms in the presence of Ca2+ were titrated with Cu2+ and activity was monitored using a factor Xa generation assay. The concentration of free Cu2+ was controlled by the presence of EDTA and Ca2+ based on the known values for Cu2+-EDTA and Ca2+-EDTA affinities. The activity regain observed for wild type factor VIII following titration with Cu2+ yielded a Kd = 11.5 ± 2.2 fM. All of the mutants tested retained a high affinity response to Cu2+, suggesting that single point mutations were not sufficient to eliminate Cu2+ binding. However, while Kd values for H1957A (10.3 ± 2.2 fM), H99A (2.1 ± 1.9 fM), H315A (6.7 ± 3.1 fM), and C310S (12.7 ± 3.4 fM) retained similar or slightly reduced affinity for Cu2+, the Kd values for C2000S (304 ± 105 fM) and H1954A (365 ± 105 fM) showed a significant reduction in Cu2+ affinity. When factor VIIIa subunits were titrated with Cu2+ and monitored by intrinsic fluorescence, only purified A1 subunit showed a saturable effect of the signal (Kd = 11.9 ± 4.9 fM), indicating that this subunit contained a high affinity Cu2+ binding site. Taken together, these results suggest that occupancy of the type 1 Cu2+ binding site in A3 contributes to the Cu2+-dependent increase in specific activity of factor VIII and that the type 1 site in A1 and/or the type 2 site contributes to HC-LC association.

Sign in / Sign up

Export Citation Format

Share Document