Ca2+-calmodulin-dependent protein kinase II potentiates store-operated Ca2+ current

Abstract

A rise in intracellular Ca2+ (Ca2+i) mediates various cellular functions ranging from fertilization to gene expression. A ubiquitous Ca2+ influx pathway that contributes significantly to the generation of Ca2+i signals, especially in non-excitable cells, is store-operated Ca2+ entry (SOCE). Consequently, the modulation of SOCE current affects Ca2+i dynamics and thus the ensuing cellular response. Therefore, it is important to define the mechanisms that regulate SOCE. Here we show that a rise in Ca2+i potentiates SOCE. This potentiation is mediated by Ca2+-calmodulin-dependent protein kinase II (CaMKII), because inhibition of endogenous CaMKII activity abrogates Ca2+i-mediated SOCE potentiation and expression of constitutively active CaMKII potentiates SOCE current independently of Ca2+i. Moreover, we present evidence that CaMKII potentiates SOCE by altering SOCE channel gating. The regulation of SOCE by CaMKII defines a novel modulatory mechanism of SOCE with important physiological consequences.

Fig. 1. Intracellular Ca2+ rise potentiates I_SOC.

A, voltage protocols (VP) used to measure I_SOC (#1) and to induce Ca²⁺ influx (#2). For VP#1, the cell was stepped to −140 mV followed by a ramp from −140 to 50 mV from a holding potential of −20 mV repeated once every 20 s. For VP#2, the cell was stepped to +40, −140, and +40 mV sequentially from a holding potential of −40 mV once every 30 s. B, time course of I_SOC activation following the BAPTA-Ion (B-I) or Ion-BAPTA (I-B) protocols. For the BAPTA-Ion protocol, oocytes were injected with BAPTA (7 nmol/oocyte) followed by ionomycin (10 μm) treatment as indicated by the line. I_SOC was measured as the average current 30–35 ms after stepping to − 140 mV in VP#1. For the Ion-BAPTA protocol, cells were treated with ionomycin and repetitively subjected to VP#2 for 10 min before I_SOC recording (open circles). La³⁺ (100 μM) was added at the end of the experiment to block I_SOC. C, summary of I_SOC levels following the BAPTA-Ion (n = 14) and Ion-BAPTA (n = 12) protocols. The data are plotted as mean ± S.E. (p = 1.6 × 10⁻⁷). D, Ca²⁺_i rise potentiates I_SOC independently of hyperpolarization-induced Ca²⁺ influx. Cells (in 5 mM Ca²⁺) were exposed to ionomycin for 5 min and then injected with BAPTA (Ion-BAPTA), and I_SOC was measured as described in B, BAPTA-Ion treatment was as described in B. The means are significantly different (p = 0.0044; n = 5).

Fig. 2. Partial store depletion fully activates SOCE.

Oocyte was loaded with Ca²⁺-Green-1-dextran (7 μM) and incubated in thapsigargin (Thaps) (1 μM) for 1 h to partially deplete intracellular Ca²⁺ stores. SOCE was measured by clamping the cell using voltage protocol number 2 (Fig. 1A) as described by Machaca and Hartzell (19). Ca²⁺-Green- 1-dextran fluorescence at +40 mV (F_Ca(+40); close squares) provides basal Ca²⁺ levels because there is minimal Ca²⁺ influx at this voltage (19). In contrast, Ca²⁺ influx through SOCE channels is induced at −140 mV, resulting in increased Ca²⁺-Green-1-dextran fluorescence (F_Ca(−140); open circles). Therefore, the difference between CG1 fluorescence at −140 and +40 mV provides a measure of SOCE. Partial store depletion with a short thapsigargin incubation (1 h) activates SOCE. Injection of IP₃ (~2 μM) leads to further Ca²⁺ release from stores as indicated by increased Ca²⁺-Green-1-dextran fluorescence at both voltages (squares and circles). However, the additional Ca²⁺ release leading to further store depletion does not enhance SOCE (open triangles). Switching the cell to Ca²⁺-free solution results in a loss of the Ca²⁺- Green-1-dextran fluorescence signal at −140 mV, confirming that the signal is because of Ca²⁺ influx. These data are representative of four similar experiments.

Fig. 3. Receptor mediated Ca2+ release levels correlate with SOCE.

Oocytes were injected with serotonin receptor 1c (5HT1c) RNA at either 10 or 30 ng/oocyte and were allowed to express for 2 days. The activity of the endogenous Ca²⁺-activated Cl⁻ currents (I_Cl1 and I_Cl1T) were recorded. I_Cl1 and I_Cl1T provide endogenous reporters of Ca²⁺ release (I_Cl1) and SOCE (I_Cl1T) (see “Results” for details). A, voltage protocol and representative current traces of the Ca²⁺-activated Cl⁻ currents. I_Cl1 is a sustained current recorded at +40 mV, whereas I_Cl1T is a transient current detected at +40 mV after the −140 mV hyperpolarization step. B and C, time course of I_Cl1 and I_Cl1T is cells injected with 10 ng (B) or 30 ng (C) 5HT1c RNA. The time course of I_Cl1 was plotted as the current at the end of the first +40-mV pulse (square in A), and that of I_Cl1T was plotted as the maximal current during the second +40-mV pulse (circle in A). C, higher 5HT1c expression results in increased levels of both I_Cl1 and I_Cl1T. D, mean I_Cl1 and I_Cl1T current levels (±S.E.) in cells expressing low (10 ng, hatched bars) and high levels (30 ng, filled bars) of 5HT1c. Increasing the expression of 5HT1c results in higher levels of I_Cl1 (indicative of Ca²⁺ release) and I_Cl1T (indicative of SOCE).

Fig. 4. CaMKIIca potentiates I_SOC.

Oocytes were injected with a constitutively active CaMKII (CaMKII^ca, 1 ng/oocyte) and allowed to express for 12–16 h. SOCE was measured using voltage protocol number 1 in Fig. 1A with the exception that the voltage was stepped to −120 mV instead of −140 mV. A, time course of I_SOC activation in control and CaMKII^ca expressing cells. B, normalized I_SOC levels (n as indicated; p = 1.1 × 10⁻⁷). C, normalized I_SOC levels from control (Con.) and CaMKII^ca-injected cells treated according to the BAPTA-Ion or Ion-BAPTA protocols as described in Fig. 1. The asterisks above the bars indicate the significantly different groups (n as indicated; p < 0.0164). D, basal CaMKII kinase activity from control and CaMKII^ca-injected oocytes measured using an in vitro kinase assay without the addition of Ca²⁺-CaM (n = 5; p = 0.0469).

Fig. 5. Inhibition of endogenous CaMKII blocks Ca²⁺ i-mediated SOCE potentiation.

Control and AIP-injected (Inj.) (10 μM) oocytes were treated according to the BAPTA-Ion and Ion-BAPTA protocols as indicated. A, normalized I_SOC levels in the different treatment groups. The asterisk indicates the only significantly different group (p < 2.1 × 10⁻⁴). B, basal CaMKII activity in oocyte lysate without AIP (Con) and with 10 and 40 μM AIP as indicated (n = 6).

Fig. 6. CaMKII potentiates I_SOC by increasing the levels of CDP.

Cells were incubated with thapsigargin (Thaps) (1 μM) in nominally Ca²⁺-free medium (50 μM) for 3 h to fully deplete Ca²⁺ stores. A subset of cells was then injected with 1 ng of CaMKII^ca RNA (Thaps-CaMK^ca) and incubated in nominally Ca²⁺-free medium for 12–16 h. A, I_SOC recorded from a representative control (Thaps) and CaMKII^ca-injected oocyte (Thaps-CaMK^ca). Cells were incubated in Ca²⁺-free solution (70 Mg²⁺) before switching to Ca²⁺-containing solution (30 Ca²⁺) as indicated by the line. SOCE was measured using voltage protocol number 1 in Fig. 1A with the exception that the voltage was stepped to −120 mV instead of −140 mV. The addition of La³⁺ to block I_SOC is also indicated. B, normalized I_SOC levels showing initial I_SOC (indicated by the asterisk in A) and maximal I_SOC at the end of the experiment (indicated by the open square and filled circle in the Thaps and Thaps-CaMK^ca groups, respectively). The average levels of CDP calculated as maximal I_SOC/initial I_SOC are also shown. Maximal I_SOC and CDP are significantly different between the two groups (p < 0.00132). C and D, current traces in response to a step voltage (−20 to −120 mV for 500 ms) (C) and a current ramp (−140 to +50 mV) (D) obtained at different time points during the experiment as indicated in A. E and F, superimposed current traces of maximal I_SOC from Thaps and Thaps-CaMKII^ca-treated cells in response to a step voltage pulse (E) and a voltage ramp (F).