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ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating.

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

Bottom Line: 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 microM.These effects were largely due to effects of ATP on the mean closed channel duration.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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-InsP(3)R-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 microM. 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 microM) 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.

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(A–D) Typical single-channel current traces of r-InsP3R-3 channels in the outer membrane of nuclei isolated from r-InsP3R-3 cRNA-injected oocytes under suboptimal [Ca2+]i, with various [ATP]i and [Mg2+]i. Arrows indicate closed channel current levels. (A) [Ca2+]i = 224 nM, [ATP]i = 0.5 mM; [Mg2+]i = 0 mM. (B) [Ca2+]i = 255 nM, [ATP]i = 0 mM; [Mg2+]i = 0 mM. (C) [Ca2+]i = 274 nM, [ATP]i = 0 mM; [Mg2+]i = 3.0 mM. (D) [Ca2+]i = 286 nM, [ATP]i = 12 μM; [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). Reduction of InsP3R channel conductance in the presence of Mg2+ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel Po of the r-InsP3R-3 channel in conditions specified in A–D. Asterisk represents P < 0.05.
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Figure 2: (A–D) Typical single-channel current traces of r-InsP3R-3 channels in the outer membrane of nuclei isolated from r-InsP3R-3 cRNA-injected oocytes under suboptimal [Ca2+]i, with various [ATP]i and [Mg2+]i. Arrows indicate closed channel current levels. (A) [Ca2+]i = 224 nM, [ATP]i = 0.5 mM; [Mg2+]i = 0 mM. (B) [Ca2+]i = 255 nM, [ATP]i = 0 mM; [Mg2+]i = 0 mM. (C) [Ca2+]i = 274 nM, [ATP]i = 0 mM; [Mg2+]i = 3.0 mM. (D) [Ca2+]i = 286 nM, [ATP]i = 12 μM; [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). Reduction of InsP3R channel conductance in the presence of Mg2+ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel Po of the r-InsP3R-3 channel in conditions specified in A–D. Asterisk represents P < 0.05.

Mentions: r-InsP3R-3 channel activities with a high Po of 0.6 (Fig. 2 A) and gating kinetics similar to those of the r-InsP3R-3 reported previously (see Mak et al. 2001, in this issue) were observed in pipet solutions containing 0.5 mM free ATP, and ∼250 nM free Ca2+. In the absence of ATP, in contrast, the r-InsP3R-3 channel had significantly lower Po of 0.2, either in the presence or absence of 3 mM Mg2+ (Fig. 2B and Fig. C). ATP did not affect the conductance of the r-InsP3R-3 channel (Fig. 2A and Fig. B). The time course of channel inactivation of the r-InsP3R-3 channel was not substantially different in the presence and absence of ATP.


ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating.

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

(A–D) Typical single-channel current traces of r-InsP3R-3 channels in the outer membrane of nuclei isolated from r-InsP3R-3 cRNA-injected oocytes under suboptimal [Ca2+]i, with various [ATP]i and [Mg2+]i. Arrows indicate closed channel current levels. (A) [Ca2+]i = 224 nM, [ATP]i = 0.5 mM; [Mg2+]i = 0 mM. (B) [Ca2+]i = 255 nM, [ATP]i = 0 mM; [Mg2+]i = 0 mM. (C) [Ca2+]i = 274 nM, [ATP]i = 0 mM; [Mg2+]i = 3.0 mM. (D) [Ca2+]i = 286 nM, [ATP]i = 12 μM; [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). Reduction of InsP3R channel conductance in the presence of Mg2+ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel Po of the r-InsP3R-3 channel in conditions specified in A–D. Asterisk represents P < 0.05.
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Related In: Results  -  Collection

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Figure 2: (A–D) Typical single-channel current traces of r-InsP3R-3 channels in the outer membrane of nuclei isolated from r-InsP3R-3 cRNA-injected oocytes under suboptimal [Ca2+]i, with various [ATP]i and [Mg2+]i. Arrows indicate closed channel current levels. (A) [Ca2+]i = 224 nM, [ATP]i = 0.5 mM; [Mg2+]i = 0 mM. (B) [Ca2+]i = 255 nM, [ATP]i = 0 mM; [Mg2+]i = 0 mM. (C) [Ca2+]i = 274 nM, [ATP]i = 0 mM; [Mg2+]i = 3.0 mM. (D) [Ca2+]i = 286 nM, [ATP]i = 12 μM; [Mg2+]i = 2.5 mM (0.5 mM total ATP; 3 mM total Mg2+). Reduction of InsP3R channel conductance in the presence of Mg2+ (C and D) is caused by permeant ion block of the channel by the divalent cation (Mak and Foskett 1998). (E) Single-channel Po of the r-InsP3R-3 channel in conditions specified in A–D. Asterisk represents P < 0.05.
Mentions: r-InsP3R-3 channel activities with a high Po of 0.6 (Fig. 2 A) and gating kinetics similar to those of the r-InsP3R-3 reported previously (see Mak et al. 2001, in this issue) were observed in pipet solutions containing 0.5 mM free ATP, and ∼250 nM free Ca2+. In the absence of ATP, in contrast, the r-InsP3R-3 channel had significantly lower Po of 0.2, either in the presence or absence of 3 mM Mg2+ (Fig. 2B and Fig. C). ATP did not affect the conductance of the r-InsP3R-3 channel (Fig. 2A and Fig. B). The time course of channel inactivation of the r-InsP3R-3 channel was not substantially different in the presence and absence of ATP.

Bottom Line: 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 microM.These effects were largely due to effects of ATP on the mean closed channel duration.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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-InsP(3)R-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 microM. 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 microM) 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.

Show MeSH
Related in: MedlinePlus