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. 1998 Dec 22;95(26):15821-5.
doi: 10.1073/pnas.95.26.15821.

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

D O Mak  1 , S McBride, J K Foskett
Affiliations

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

D O Mak et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A 1999 Mar 16;96(6):3330

Abstract

Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

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Figures

Figure 1
Figure 1
[Ca2+]i dependence of the IP3R in the presence of 10 μM IP3. (a) Typical single-channel current traces of the IP3R at various [Ca2+]i. Arrows indicate closed-channel current level in all traces. (b) Dependence on [Ca2+]i of mean closed-channel duration τc and open-channel duration τo of the IP3R. (c) Dependence of IP3R Po on [Ca2+]i. Experiments were performed with different Ca2+ chelators (□, BAPTA; ▵, 5,5′-dibromo BAPTA, ○, ATP) in the pipette solutions. Bath solution contained 1.5 μM Ca2+ and 0.5 mM ATP. Numerical labels indicate calculated concentrations (in μM) of free Ca2+ chelators present in the pipette solutions for the corresponding data points. The solid curve is the Hill equation fit for the biphasic [Ca2+]i effect on IP3R Po. (d) IP3R Po vs. [Ca2+]i in various bath solutions. Small open symbols: 0.8 μM luminal [Ca2+], 0.5 mM ATP; large open symbols: 0.2 μM luminal [Ca2+], 0 ATP; solid symbols: 0.2 μM luminal [Ca2+], 0.5 mM ATP. Convention for symbols and the solid curve are the same as in c.
Figure 2
Figure 2
IP3-concentration dependence of the [Ca2+]i sensitivity of the IP3R. Different symbols denote data for various IP3 concentrations as tabulated. Data points for 10 μM IP3 were combined from experiments using various bath solutions with the same pipette [Ca2+]. Data points for other concentrations of IP3 were obtained in bath solutions containing 220 nM Ca2+ and no ATP. The curves are Hill equation fits using Eq. 1, with Kinh varying with IP3 concentration with values as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of IP3 concentration, with the values tabulated in the graph. Numerical labels indicate calculated concentrations (in μM) of free Ca2+ chelator BAPTA present in pipette solutions used for the corresponding data points.
Figure 3
Figure 3
(a) Dependence on IP3 concentration of Kinh as derived from Fig. 2. The curve is a simple Hill equation fit. (b) Bell-shaped normalized Po-vs.-[Ca2+]i curves calculated by using Eqs. 1 and 2 for various low concentrations of IP3 (dotted curve, 10 nM IP3 with maximum Po of 0.09; dashed curve, 15 nM IP3 with maximum Po of 0.49; and solid curve, 20 nM IP3 with maximum Po of 0.71). (c) Theoretical Po (calculated by using the model) vs. IP3 concentration at various [Ca2+]i, as labeled. Dashed curves, [Ca2+]i ≤ 1 μM; thin solid curves, [Ca2+]i ≥ 10 μM.
Figure 4
Figure 4
Calculated Po vs. [Ca2+]i and IP3 concentration according to our model. (a) Projection showing Po-vs.-IP3 concentration curves at various activating [Ca2+]i. (b) Projection showing Po-vs.-[Ca2+]i curves at various IP3 concentrations. (c) Projection showing Po vs. IP3 concentration curves at various inhibitory [Ca2+]i.
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