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Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors.

Mak DO, McBride S, Foskett JK - J. Gen. Physiol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) 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 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) 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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Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the Hill equation (), with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph.
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Figure 4: Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the Hill equation (), with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph.

Mentions: To examine specifically the effects of [Ca2+]i on r-InsP3R-3 channel gating, a functionally saturating concentration of InsP3 (10 μM) was applied to the cytoplasmic (pipet) side of the channel to stimulate it fully at all experimental [Ca2+]i (Fig. 2). At [Ca2+]i corresponding to resting levels in cells (10–100 nM), the Po of the r-InsP3R-3 channel was moderate (<0.5; Fig. 4), with the channel evidently active (Fig. 2 A). The Po increased to ∼0.8 when [Ca2+]i was raised from 100 nM to 1 μM, which was associated with decreasing mean closed duration (τc) (Fig. 3 and Fig. 4). Between [Ca2+]i of 1 and 25 μM, Po remained high (∼0.8; Fig. 3 and Fig. 4), with the channel exhibiting long sustained bursts of activities lasting up to several seconds, during which it only closed briefly (Fig. 2 C). As [Ca2+]i was increased beyond 25 μM, Po dropped precipitously, as a result of an increase in τc to >200 ms (Fig. 3 and Fig. 4). Within the more than three orders of magnitude range of [Ca2+]i examined (24.7 nM–82.8 μM), the mean open duration (τo) of the r-InsP3R-3 channel lay within a narrow range (4–16 ms) with no systematic dependence on [Ca2+]i (Fig. 3 A). In contrast, τc changed about two orders of magnitude (from 3 to 210 ms; Fig. 3 B) over the same range of [Ca2+]i, accounting for most of the strong dependence of channel Po on [Ca2+]i (Fig. 4).


Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors.

Mak DO, McBride S, Foskett JK - J. Gen. Physiol. (2001)

Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the Hill equation (), with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233662&req=5

Figure 4: Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the Hill equation (), with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph.
Mentions: To examine specifically the effects of [Ca2+]i on r-InsP3R-3 channel gating, a functionally saturating concentration of InsP3 (10 μM) was applied to the cytoplasmic (pipet) side of the channel to stimulate it fully at all experimental [Ca2+]i (Fig. 2). At [Ca2+]i corresponding to resting levels in cells (10–100 nM), the Po of the r-InsP3R-3 channel was moderate (<0.5; Fig. 4), with the channel evidently active (Fig. 2 A). The Po increased to ∼0.8 when [Ca2+]i was raised from 100 nM to 1 μM, which was associated with decreasing mean closed duration (τc) (Fig. 3 and Fig. 4). Between [Ca2+]i of 1 and 25 μM, Po remained high (∼0.8; Fig. 3 and Fig. 4), with the channel exhibiting long sustained bursts of activities lasting up to several seconds, during which it only closed briefly (Fig. 2 C). As [Ca2+]i was increased beyond 25 μM, Po dropped precipitously, as a result of an increase in τc to >200 ms (Fig. 3 and Fig. 4). Within the more than three orders of magnitude range of [Ca2+]i examined (24.7 nM–82.8 μM), the mean open duration (τo) of the r-InsP3R-3 channel lay within a narrow range (4–16 ms) with no systematic dependence on [Ca2+]i (Fig. 3 A). In contrast, τc changed about two orders of magnitude (from 3 to 210 ms; Fig. 3 B) over the same range of [Ca2+]i, accounting for most of the strong dependence of channel Po on [Ca2+]i (Fig. 4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) 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 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) 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.

Show MeSH
Related in: MedlinePlus