Single Channel Function of Recombinant Type-1 Inositol 1,4,5-Trisphosphate Receptor Ligand Binding Domain Splice Variants

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.

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

Biophysical Journal ◽

10.1016/s0006-3495(00)76391-0 ◽

2000 ◽

Vol 79 (3) ◽

pp. 1388-1399 ◽

Cited By ~ 72

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

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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

Biophysical Journal ◽

10.1016/j.bpj.2010.12.1584 ◽

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

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Single Channel Function of Inositol 1,4,5-trisphosphate Receptor Type-1 and -2 Isoform Domain-Swap Chimeras

The Journal of General Physiology ◽

10.1085/jgp.200208718 ◽

2003 ◽

Vol 121 (5) ◽

pp. 399-411 ◽

Cited By ~ 9

Author(s):

Jorge Ramos ◽

Wonyong Jung ◽

Josefina Ramos-Franco ◽

Gregory A. Mignery ◽

Michael Fill

Keyword(s):

Lipid Bilayers ◽

Single Channel ◽

Wild Type ◽

Structural Determinants ◽

Domain Swap ◽

Channel Function ◽

Recombinant Type ◽

Ligand Regulation

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.

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Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

Biochemical Journal ◽

10.1042/bj3460275 ◽

2000 ◽

Vol 346 (2) ◽

pp. 275-280 ◽

Cited By ~ 20

Author(s):

Sara VANLINGEN ◽

Henk SIPMA ◽

Patrick DE SMET ◽

Geert CALLEWAERT ◽

Ludwig MISSIAEN ◽

...

Keyword(s):

Ligand Binding ◽

Recombinant Proteins ◽

Ip3 Receptor ◽

Binding Characteristics ◽

Binding Domains ◽

Receptor Isoforms ◽

Inositol 1,4,5 Trisphosphate ◽

Adenophostin A ◽

Trisphosphate Receptor

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.

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The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3

Biochemical Journal ◽

10.1042/bj20030599 ◽

2004 ◽

Vol 377 (2) ◽

pp. 299-307 ◽

Cited By ~ 71

Author(s):

Tomohiro NAKAYAMA ◽

Mitsuharu HATTORI ◽

Keiko UCHIDA ◽

Takeshi NAKAMURA ◽

Yoko TATEISHI ◽

...

Keyword(s):

Ligand Binding ◽

Caspase 3 ◽

Fluorescent Protein ◽

Binding Domain ◽

Ligand Binding Domain ◽

Physiological Significance ◽

Regulatory Domain ◽

Inositol 1,4,5 Trisphosphate ◽

Trisphosphate Receptor ◽

In Cells

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is an intracellular Ca2+ channel protein that plays crucial roles in generating complex Ca2+ signalling patterns. IP3R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP3R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP3R1 or IP3R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells’ responsiveness to an IP3-generating agonist ATP and Ca2+ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP–IP3R1-casp) or the channel domain alone (GFP–IP3R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca2+ concentration. Interestingly, store-operated (−like) Ca2+ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca2+ stores of cells expressing GFP–IP3R1-casp or GFP–IP3R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca2+. We therefore propose that the channel domain of IP3R1 tends to remain open and that the large regulatory domain of IP3R1 is necessary to keep the channel domain closed. Thus cleavage of IP3R1 by caspase 3 may contribute to the increased cytosolic Ca2+ concentration often observed in cells undergoing apoptosis. Finally, GFP–IP3R1-casp or GFP–IP3R1-ES can be used as a novel tool to deplete intracellular Ca2+ stores.

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The complex regulatory function of the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor

Cell Calcium ◽

10.1016/j.ceca.2007.04.005 ◽

2008 ◽

Vol 43 (1) ◽

pp. 17-27 ◽

Cited By ~ 25

Author(s):

Benoit Devogelaere ◽

Leen Verbert ◽

Jan B. Parys ◽

Ludwig Missiaen ◽

Humbert De Smedt

Keyword(s):

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Regulatory Function ◽

Inositol 1,4,5 Trisphosphate ◽

Trisphosphate Receptor

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Association of the Inositol (1,4,5)-Trisphosphate Receptor Ligand Binding Site with Phosphatidylinositol (4,5)-Bisphosphate and Adenophostin A

Molecular Cell Biology Research Communications ◽

10.1006/mcbr.2000.0208 ◽

2000 ◽

Vol 3 (3) ◽

pp. 153-158 ◽

Cited By ~ 17

Author(s):

Lyuba Glouchankova ◽

U.Murali Krishna ◽

Barry V.L. Potter ◽

J.Russell Falck ◽

Ilya Bezprozvanny

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Ligand Binding Site ◽

Receptor Ligand ◽

Inositol 1,4,5 Trisphosphate ◽

Adenophostin A ◽

Trisphosphate Receptor

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

The Journal of General Physiology ◽

10.1085/jgp.117.5.435 ◽

2001 ◽

Vol 117 (5) ◽

pp. 435-446 ◽

Cited By ~ 102

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m109.006452 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16156-16163 ◽

Cited By ~ 30

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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