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

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.

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 τ_c and open-channel duration τ_o of the IP₃R. (c) Dependence of IP₃R P_o 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 P_o. (d) IP₃R P_o 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

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 K_inh varying with IP₃ concentration with values as listed in the graph, whereas P_max, K_act, H_act, and H_inh 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

(a) Dependence on IP₃ concentration of K_inh as derived from Fig. 2. The curve is a simple Hill equation fit. (b) Bell-shaped normalized P_o-vs.-[Ca²⁺]_i curves calculated by using Eqs. 1 and 2 for various low concentrations of IP₃ (dotted curve, 10 nM IP₃ with maximum P_o of 0.09; dashed curve, 15 nM IP₃ with maximum P_o of 0.49; and solid curve, 20 nM IP₃ with maximum P_o of 0.71). (c) Theoretical P_o (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

Calculated P_o vs. [Ca²⁺]_i and IP₃ concentration according to our model. (a) Projection showing P_o-vs.-IP₃ concentration curves at various activating [Ca²⁺]_i. (b) Projection showing P_o-vs.-[Ca²⁺]_i curves at various IP₃ concentrations. (c) Projection showing P_o vs. IP₃ concentration curves at various inhibitory [Ca²⁺]_i.