Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

Abstract

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% +/- 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 +/- 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Figure 1

Reversible binding of ¹²⁵I-calmodulin to pure InsP₃ receptors in the absence and presence of Ca²⁺. (A) The time course of specific binding of ¹²⁵I-calmodulin (3 nM) to SPA-InsP₃ receptor beads is shown, first in nominally Ca²⁺-free medium ([Ca²⁺]_m ≈ 2 nM), then after addition of Ca²⁺ ([Ca²⁺]_m ≈ 30 μM), and finally after addition of EGTA (40 mM) to restore [Ca²⁺]_m to ≈2 nM. The main panel illustrates results from a single experiment, representative of three. Equilibrium binding of ¹²⁵I-calmodulin in Ca²⁺-free medium (n = 12), Ca²⁺-containing medium (n = 12), and after addition of EGTA (n = 3) are shown in the histogram (means ± SEM). (B) The stimulatory effect of [Ca²⁺]_m on specific ¹²⁵I-calmodulin binding is plotted as a percentage of the maximal effect, which was obtained when [Ca²⁺]_m was ≈30 μM. The 0 and 100% values were both derived by extrapolation of the curves. Results are means ± SEM of three independent experiments. (C) Dissociation of ¹²⁵I-calmodulin from InsP₃ receptors is shown after addition of calmodulin (50 μM) to SPA-InsP₃ receptor beads that had equilibrated (3 h) with ¹²⁵I-calmodulin (3 nM) in the absence (•, [Ca²⁺]_m ≈ 2 nM) or presence (○, [Ca²⁺]_m ≈ 30 μM) of Ca²⁺. Results are plotted on a semilogarithmic scale and are representative of three independent experiments. In Ca²⁺-free medium, 45% ± 5% of the ¹²⁵I-calmodulin dissociated with a half-time (t½) = 474 ± 156 s, and the remainder with a t½ = 143 ± 29 min; the comparable numbers in Ca²⁺-containing medium were t½ = 180 ± 48 s (44% ± 2%) and 74 ± 9 min.

Figure 2

Equilibrium binding of ¹²⁵I-calmodulin to pure InsP₃ receptors in the absence and presence of Ca²⁺. SPA-InsP₃ receptor beads were incubated with ¹²⁵I-calmodulin (3 nM) in the presence of the indicated concentrations of unlabeled calmodulin, first in Ca²⁺-free medium (•, [Ca²⁺]_m ≈ 2 nM) and then after addition of Ca²⁺ (○, [Ca²⁺]_m ≈ 30 μM). C denotes the control. Results (means ± SEM) are from three independent experiments.

Figure 3

Calmodulin inhibits [³H]InsP₃ binding to InsP₃ receptors. (A) Effects of calmodulin in Ca²⁺-free medium on equilibrium binding of [³H]InsP₃ (3 nM) to cerebellar membranes (○, typical results from one of three independent preparations) or to pure InsP₃ receptors on SPA beads (•, means ± SEM of three independent receptor purifications). Results are shown as percentages of the maximal inhibition obtained in the presence of a saturating concentration of calmodulin (derived by extrapolation of the binding curve to infinite calmodulin concentration). (Inset) Typical effects of calmodulin on specific binding of [³H]InsP₃ to pure InsP₃ receptors from two preparations, showing the differences in the maximal inhibition caused by calmodulin. (B) Equilibrium competition binding curves are shown for specific [³H]InsP₃ binding to InsP₃ receptors in Ca²⁺-free medium in the absence of calmodulin (•) and then after addition of a submaximal concentration of calmodulin (3 μM) (○). Results are the means ± SEM of three independent experiments.

Figure 4

Calmodulin inhibits InsP₃-evoked Ca²⁺ release from cerebellar microsomes. Cerebellar microsomes were loaded with ⁴⁵Ca²⁺ in the absence (○) or presence (•) of 10 μM calmodulin before addition of the indicated concentrations of InsP₃ in the continued presence or absence of calmodulin. The results (means ± SEM of 3–17 independent experiments, each performed in triplicate) show the amount of Ca²⁺ released during the 45-s incubation with InsP₃.

Figure 5

Calmodulin inhibition of [³H]InsP₃ binding is independent of Ca²⁺. Results (means ± SEM of three independent experiments) show the effect of varying [Ca²⁺]_m on the specific binding of [³H]InsP₃ (3 nM) to receptor beads in the absence (•) and presence (○) of a submaximal concentration of calmodulin (3 μM).

Figure 6

Interactions between calmodulin and neuronal InsP₃ receptors. (A) Predicted structure of a single subunit of the type 1 InsP₃ receptor. Our results establish that both calmodulin and Ca²⁺–calmodulin bind with the same affinity to a site on the InsP₃ receptor to decrease its affinity for InsP₃; the exact location of this site 1 is unknown. Another calmodulin-binding site (site 2) within the modulatory domain of the receptor binds only Ca²⁺–calmodulin (27) and, as the helical wheel representation demonstrates, that site has the basic amphipathic helical structure found in other Ca²⁺–calmodulin-binding proteins (42). Within the helical wheel, basic residues (Arg, His, Lys) are denoted by •, and hydrophobic residues (Ile, Ala, Trp, Val, Leu) by ○. (B) Both neurogranin (postsynaptic) and neuromodulin (presynaptic) are exclusively neuronal and release their bound calmodulin after either an increase in cytosolic Ca²⁺ concentration or phosphorylation by protein kinase C (PKC). CaMKII binds Ca²⁺–calmodulin, which triggers autophosphorylation causing the calmodulin to remain bound after the Ca²⁺ concentration has returned to its resting level. The ensuing changes in cytosolic calmodulin concentration will regulate binding of InsP₃ to its receptor irrespective of the prevailing cytosolic Ca²⁺ concentration.