scholarly journals Atp Regulation of Recombinant Type 3 Inositol 1,4,5-Trisphosphate Receptor Gating

2001 ◽  
Vol 117 (5) ◽  
pp. 447-456 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Sean McBride ◽  
J. Kevin Foskett
Keyword(s):  
Free Acid ◽  
Second Messengers ◽  
Hill Coefficient ◽  
Open Probability ◽  
Isoform Diversity ◽  
Recombinant Type ◽  
Inositol 1,4,5 Trisphosphate ◽  
Patch Clamp Electrophysiology ◽  
Type 3

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 (<1 μM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.

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 Unitary Ca2+ current through recombinant type 3 InsP3 receptor channels under physiological ionic conditions

2010 ◽  
Vol 136 (6) ◽  
pp. 687-700 ◽  
Author(s):  
Horia Vais ◽  
J. Kevin Foskett ◽  
Don-On Daniel Mak
Keyword(s):  
Open Probability ◽  
Physiological Processes ◽  
Perfusion Solution ◽  
Recombinant Type ◽  
Extracellular Stimuli ◽  
Rapid Perfusion ◽  
Patch Clamp Electrophysiology ◽  
Active Channels ◽  
Type 3 ◽  
Insp3 Receptor

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca2+ into the cytoplasm upon binding InsP3, generating and modulating intracellular Ca2+ 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 Ca2+ current (iCa) passing through an open InsP3R 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 InsP3R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of iCa through an InsP3R 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 InsP3R 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 InsP3R 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 InsP3-liganded InsP3R channels, and they confirm that Ca2+ released by an open InsP3R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca2+-induced Ca2+ release.

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 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 Atp-Dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating

2001 ◽  
Vol 117 (4) ◽  
pp. 299-314 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Sean McBride ◽  
J. Kevin Foskett
Keyword(s):  
Single Channel ◽  
Free Acid ◽  
Channel Gating ◽  
Xenopus Laevis Oocyte ◽  
Open Probability ◽  
Release Channel ◽  
Gating Kinetics ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinetics Of ◽  
In Cells

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is a ligand-gated intracellular Ca2+ release channel that plays a central role in modulating cytoplasmic free Ca2+ concentration ([Ca2+]i). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP3R that is structurally different from InsP3 and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP3R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP3R activated by either AdA or InsP3 have identical channel conductance properties. Furthermore, AdA, like InsP3, activates the channel by tuning Ca2+ inhibition of gating. However, gating of the AdA-liganded InsP3R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP3-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP3 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 InsP3R 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 InsP3 in the presence or absence of ATP. Also, the higher functional affinity of InsP3R for AdA than for InsP3 is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP3R 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 Ca2+ release events in cells. Comparisons of single-channel gating kinetics of the InsP3R activated by InsP3, AdA, and its analogues also identify molecular elements in InsP3R ligands that contribute to binding and activation of channel gating.

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 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.

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