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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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Comparison of the Ca2+ dependencies of Po of r-InsP3R-3 and X-InsP3R-1 in 10 μM InsP3 and 0.5 mM ATP. Open circles represent data for r-InsP3R-3 from this study, fitted with the solid curve; closed circles represent data for X-InsP3R-1 taken from (Mak et al. 1998), fitted with the dashed curve. The curves are calculated using the Hill equation () with the tabulated parameters. Higher affinity and lack of cooperativity of the Ca2+ activation sites of the type 3 channel endow it with high gain IICR and low gain CICR. In contrast, lower affinity and presence of cooperativity of the Ca2+ activation sites of the type 1 channel confer low gain IICR and high gain CICR. Under resting [Ca2+]i, low levels of stimulation will trigger release of Ca2+ by IICR from the type 3 channel, which in turn will trigger further release by CICR from the type 1 channel.
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Figure 6: Comparison of the Ca2+ dependencies of Po of r-InsP3R-3 and X-InsP3R-1 in 10 μM InsP3 and 0.5 mM ATP. Open circles represent data for r-InsP3R-3 from this study, fitted with the solid curve; closed circles represent data for X-InsP3R-1 taken from (Mak et al. 1998), fitted with the dashed curve. The curves are calculated using the Hill equation () with the tabulated parameters. Higher affinity and lack of cooperativity of the Ca2+ activation sites of the type 3 channel endow it with high gain IICR and low gain CICR. In contrast, lower affinity and presence of cooperativity of the Ca2+ activation sites of the type 1 channel confer low gain IICR and high gain CICR. Under resting [Ca2+]i, low levels of stimulation will trigger release of Ca2+ by IICR from the type 3 channel, which in turn will trigger further release by CICR from the type 1 channel.

Mentions: The activating Ca2+ binding sites of the r-InsP3R-3 had a half-maximal activating [Ca2+]i, Kact, of 77 nM and Hact of ∼1. These values contrast markedly with those obtained for the X-InsP3R-1 (Mak et al. 1998), where Kact ∼ 210 nM and Hact ∼ 2 (Fig. 6). Thus, in addition to a higher intrinsic sensitivity of the activating sites for Ca2+, the type 3 receptor lacks the apparent cooperativity among these sites that is observed in the type 1 receptor. This result agrees well with the flatter Ca2+ dependence of InsP3-induced Ca2+ release observed in B cells genetically engineered to express only InsP3R-3 compared with that observed in cells expressing InsP3R-1 only (Miyakawa et al. 1999). In light of the similarities between the two isoforms in their permeation and gating properties (Mak et al. 2000), and Ca2+ inhibition and regulation by InsP3 (this study), these differences in Ca2+ activation of the InsP3R channels represent the major distinguishing features between the two channels in our studies. Importantly, neither Kact nor Hact is affected by [InsP3] in either channel.


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)

Comparison of the Ca2+ dependencies of Po of r-InsP3R-3 and X-InsP3R-1 in 10 μM InsP3 and 0.5 mM ATP. Open circles represent data for r-InsP3R-3 from this study, fitted with the solid curve; closed circles represent data for X-InsP3R-1 taken from (Mak et al. 1998), fitted with the dashed curve. The curves are calculated using the Hill equation () with the tabulated parameters. Higher affinity and lack of cooperativity of the Ca2+ activation sites of the type 3 channel endow it with high gain IICR and low gain CICR. In contrast, lower affinity and presence of cooperativity of the Ca2+ activation sites of the type 1 channel confer low gain IICR and high gain CICR. Under resting [Ca2+]i, low levels of stimulation will trigger release of Ca2+ by IICR from the type 3 channel, which in turn will trigger further release by CICR from the type 1 channel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Comparison of the Ca2+ dependencies of Po of r-InsP3R-3 and X-InsP3R-1 in 10 μM InsP3 and 0.5 mM ATP. Open circles represent data for r-InsP3R-3 from this study, fitted with the solid curve; closed circles represent data for X-InsP3R-1 taken from (Mak et al. 1998), fitted with the dashed curve. The curves are calculated using the Hill equation () with the tabulated parameters. Higher affinity and lack of cooperativity of the Ca2+ activation sites of the type 3 channel endow it with high gain IICR and low gain CICR. In contrast, lower affinity and presence of cooperativity of the Ca2+ activation sites of the type 1 channel confer low gain IICR and high gain CICR. Under resting [Ca2+]i, low levels of stimulation will trigger release of Ca2+ by IICR from the type 3 channel, which in turn will trigger further release by CICR from the type 1 channel.
Mentions: The activating Ca2+ binding sites of the r-InsP3R-3 had a half-maximal activating [Ca2+]i, Kact, of 77 nM and Hact of ∼1. These values contrast markedly with those obtained for the X-InsP3R-1 (Mak et al. 1998), where Kact ∼ 210 nM and Hact ∼ 2 (Fig. 6). Thus, in addition to a higher intrinsic sensitivity of the activating sites for Ca2+, the type 3 receptor lacks the apparent cooperativity among these sites that is observed in the type 1 receptor. This result agrees well with the flatter Ca2+ dependence of InsP3-induced Ca2+ release observed in B cells genetically engineered to express only InsP3R-3 compared with that observed in cells expressing InsP3R-1 only (Miyakawa et al. 1999). In light of the similarities between the two isoforms in their permeation and gating properties (Mak et al. 2000), and Ca2+ inhibition and regulation by InsP3 (this study), these differences in Ca2+ activation of the InsP3R channels represent the major distinguishing features between the two channels in our studies. Importantly, neither Kact nor Hact is affected by [InsP3] in either channel.

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