An ultrasensitive Ca2+/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling

Abstract

The strength of hippocampal synapses can be persistently increased by signals that activate Ca2+/calmodulin-dependent protein kinase II (CaMKII). This CaMKII-dependent long-term potentiation is important for hippocampal learning and memory. In this work we show that CaMKII exhibits an intriguing switch-like activation that likely is important for changes in synaptic strength. We found that autophosphorylation of CaMKII by itself showed a steep dependence on Ca2+ concentration [Hill coefficient (nH) approximately 5]. However, an even steeper Ca2+ dependence (nH approximately 8) was observed when autophosphorylation is balanced by the dephosphorylation activity of protein phosphatase 1 (PP1). This autophosphorylation-dephosphorylation switch was found to be reversible because PP1 dephosphorylates CaMKII when Ca2+ is lowered to a basal level. The switch-like response of a CaMKII-PP1 system suggests that CaMKII and PP1 may function together as a simple molecular device that specifically translates only strong Ca2+ signals into all-or-none potentiation of individual hippocampal synapses.

Fig. 1.

CaMKII autophosphorylation is cooperative to Ca²⁺. Plotted is the CaMKII autophosphorylation level (indicated by the maximum CaMKII autonomy) after 5 min versus [Ca²⁺] [error bars are ± SD; the number of autonomous activity values determined (n) was 2]. The solid line is the best fit to the Hill equation (Eq. 1). Here, [CaMKII] = 0.2 μM. All shown Ca²⁺ titration data are representative data obtained from at least two, and often three or more, data sets collected at each condition.

Fig. 2.

Dissecting the molecular basis of Ca²⁺ ultrasensitivity. (A) Lack of significant cooperativity in CaM activation of CaMKII. Plotted is the Ca²⁺-dependent CaMKII activity versus [CaM] (±SD, n = 2). (B) Modest Ca²⁺ ultrasensitivity (n_H ≈ 3) caused by cooperative binding of Ca²⁺ to CaM. Plotted is the CaM-dependent CaMKII activity versus [Ca²⁺] (±SD, n = 2). (C) Greater Ca²⁺ ultrasensitivity (n_H ≈ 5) induced by the dual requirement of CaM for autophosphorylation. Plotted is the maximum autonomy versus [Ca²⁺](±SD, n = 2). Experiments were performed at 0.2 μM (triangle, solid black line), 1.0μM (diamond, dotted line), and 5.0 μM (square, solid gray line) CaMKII.

Fig. 3.

A CaMKII-PP1 system is a reversible switch. (A) Plotted is the CaMKII autophosphorylation level (or percent of maximum CaMKII autonomy) at equilibrium versus [Ca²⁺] (±SD, n = 2). Open squares and black diamonds represent experiments initiated with dephosphorylated and phosphorylated CaMKII, respectively. The solid line is the best fit of the initially dephosphorylated CaMKII data to the Hill equation (Eq. 1). Here, [CaMKII] = 1 μM and [PP1] = 1.25 μM. (B-D) Role of varying [CaMKII] and [PP1] on the CaMKII-PP1 switch. Plotted is the CaMKII autonomy at equilibrium versus [Ca²⁺]. Squares, diamonds, and triangles represent experiments performed with 0.5, 1.25, and 2.5 μM PP1, respectively. The solid gray, dashed, and solid black lines represent the best fit of the 0.5, 1.25, and 2.5 μM PP1 data, respectively, to the Hill equation (Eq. 1). B-D show data at 0.2, 1, and 5 μM CaMKII, respectively.

Fig. 4.

Characterization of CaMKII dephosphorylation by PP1. (A) Michaelis-Menton plot of CaMKII dephosphorylation by PP1. Plotted is the velocity of dephosphorylation versus [CaMKII] (±SD, n = 2). The line represents the best fit to the Michaelis-Menton equation. (B) Time course of CaMKII dephosphorylation in the presence and absence of Ca²⁺. Plotted is the CaMKII autonomy versus time (±SD, n = 2).

Fig. 5.

How a CaMKII-PP1 switch facilitates specificity in synaptic signaling. Shown are the molecular events causing the CaMKII-PP1 switch (A) and the effect of these events on the Ca²⁺ dependence of CaMKII autophosphorylation (B). Plotted in the graph is the percent CaMKII autophosphorylation versus the range of [Ca²⁺] in dendritic spines. If CaMKII activation were noncooperative, the activation curve would encompass the entire range of physiological Ca²⁺ values [dashed blue line and (0)]. Ultrasensitivity in CaMKII activation is imposed by both the cooperative binding of Ca²⁺ to CaM [dashed green line and (1)] and the dual requirement of CaM for autophosphorylation [dashed orange line and (2)]. Competitive dephosphorylation by PP1 further enhances the switch-like behavior and increases the Ca_1/2²⁺ value to the physiological [Ca²⁺] corresponding to LTP [solid red line and (3)]. The Ca²⁺ activation regions corresponding to LTP and LTD were determined based on the Ca²⁺ level in dendritic spines under depolarizing and resting potentials, respectively (3).