Ca²⁺-Calmodulin-dependent Protein Kinase II Potentiates Store-operated Ca²⁺ Current

Abstract

A rise in intracellular Ca²⁺_i (Ca²⁺) 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.

A rise in intracellular Ca²⁺ (Ca²⁺_i)¹ mediates various cellular responses ranging from exocytosis to cell death, often in the same cell. This is possible because of the specificity encoded in the spatial, temporal, and amplitude features of Ca²⁺ signals, which are generated because of either Ca²⁺ release from intracellular stores or Ca²⁺ influx from the extracellular space. The interplay between these two Ca²⁺ sources results in specific Ca²⁺_i dynamics, which lead to a particular cellular response. A ubiquitous Ca²⁺ influx pathway activated downstream of phospholipase- coupled receptors is store-operated Ca²⁺ entry (SOCE) (1). SOCE is important for various cellular functions including store refilling (2), regulation of exocytosis (3), sperm capacitation (4), and T-cell activation (5, 6). Because SOCE contributes significantly to the generation of Ca²⁺_i dynamics, it is important in specifying the ensuing cellular response. SOCE is defined as Ca²⁺ influx activated in response to intracellular Ca²⁺ store depletion. Although, store depletion is the primary required signal for SOCE activation, additional distinct mechanisms modulate SOCE activity (1). Characterizing such SOCE modulators is crucial to understanding the mechanisms by which SOCE contributes to defining specific cellular responses. In this paper, we show that both Ca²⁺_i and Ca²⁺-calmodulindependent protein kinase II (CaMKII) positively modulate SOCE current (I_SOC).

Both intracellular and extracellular Ca²⁺ have been shown to modulate SOCE activity in a complex fashion. This is best characterized for the Ca²⁺ release-activated Ca²⁺ current (I_CRAC) (7), which is the first described SOCE current. Hallmarks of I_CRAC include inward rectification, an ionic selectivity sequence of Ca²⁺ > Ba²⁺ > Mn²⁺ >> Na⁺, and a small unitary Ca²⁺ conductance estimated to be ~20 femtoSiemens (8, 9). Intracellular Ca²⁺ has been shown to negatively regulate I_SOC by two mechanisms. 1) Fast Ca²⁺-dependent inactivation is due to inhibition of I_SOC, arguably following Ca²⁺ binding to an intracellular site (8, 10). 2) Slow Ca²⁺-dependent inactivation is due in part to store refilling but also has a store-independent component that is poorly understood (11). In contrast, extracellular Ca²⁺ (Ca²⁺_o) potentiates I_SOC through a process termed Ca²⁺-dependent potentiation (CDP). CDP is attributed to a positive effect of extracellular Ca²⁺ on I_SOC (12, 13) and probably results from Ca²⁺ binding to an extracellular site on the channel because it can be replicated by Ni²⁺ (12). Ni²⁺ does not permeate SOCE channels but potentiates the Ca²⁺ current through SOCE channels (12). Furthermore, CDP alters channel gating and not permeation (12), because current inactivation (which is dependent on permeation (8)) remains unchanged during CDP.

In this paper, we describe a novel positive regulation of I_SOC by intracellular Ca²⁺ through CaMKII in Xenopus oocytes. Xenopus oocyte SOCE current has similar characteristics to I_CRAC including high Ca²⁺ selectivity, inward rectification (14), and as shown here extracellular Ca²⁺-dependent potentiation. Allowing a Ca²⁺_i rise during SOCE activation results in larger I_SOC. Furthermore, expression of a constitutively active CaMKII (CaMKII^ca) also leads to enhancement of I_SOC. CaMKII potentiates I_SOC by dramatically increasing the levels of CDP. Because CDP affects SOCE channel gating, our data argue that CaMKII potentiates SOCE by altering gating. CaMKII-mediated potentiation of I_SOC has important physiological consequences because store depletion is physiologically accompanied by a rise in Ca²⁺_i. Because CaMKII is able to decode Ca²⁺_i dynamics into different levels of enzyme activity (15), CaMKII-mediated potentiation of I_SOC provides a mechanism for Ca²⁺ to regulate its own entry into the cell depending on the levels and kinetics of Ca²⁺ release following receptor activation.

EXPERIMENTAL PROCEDURES

Oocyte and Electrophysiological Methods

Xenopus laevis oocytes were prepared as described previously (16). SOCE was activated by the depletion of intracellular Ca²⁺ stores with either ionomycin (10 μM) or thapsigargin (1 μm for ≥3 h unless otherwise indicated). Oocytes were injected with 7 nmol of BAPTA to buffer the Ca²⁺_i rise and block the endogenous Ca²⁺-activated Cl⁻ current (I_Cl,Ca) that would otherwise mask the SOCE current. Assuming an oocyte volume of 1 μl, this would result in a final concentration of ~ 7 mm BAPTA that completely blocks I_Cl,Ca. Oocytes were voltage-clamped with two microelectrodes by the use of a GeneClamp 500 (Axon Instruments). Electrodes were filled with 3 m KCl and had resistances of 0.5–2 megaohms. Voltage stimulation and data acquisition were controlled using a pClamp8 (Axon Instruments). Current data were filtered at 10 kHz, digitized, and analyzed using Clampfit9.0 (Axon Instruments) and Origin® software (Microcal Software Inc.). I_SOC was typically measured in 30 Ca²⁺ solution (in mm: 55 NaCl, 30 CaCl₂, 10 Hepes, pH 7.4), and Ca²⁺-free solution in I_SOC-recording experiments was 70 Mg²⁺ (in mm: 70 MgCl₂, 10 Hepes, pH 7.4). I_SOC time course is plotted as the mean current 30–35 ms after the voltage step, and the leak current at the beginning of the experiment was subtracted.

Ca²⁺ activated Cl⁻ currents were recorded in Ringer solution (in mm: 123 NaCl, 2.5 KCl, 1.8 CaCl₂, 18 MgCl₂, 10 Hepes, pH 7.4) or Ca²⁺-free Ringer solution that had the same composition as Ringer solution with the exception that CaCl₂ was omitted, MgCl₂ increased to 5 mm, and 0.1 mm EGTA was added.

cRNA for injection into oocytes was transcribed in vitro using the mMessage mMachine SP6 transcription kit (Ambion). Both pGEM3– 5HT1c and pSP-CaMKII(T286D) were linearized with EcoRI and transcribed with SP6 polymerase.

Ca²⁺ Imaging

Xenopus oocytes were injected with ~ 7.6 μm Ca²⁺- Green-1 coupled to 70 kDa of dextran and voltage-clamped as described above. The dye was allowed at least 30 min to equilibrate within the oocyte. Imaging experiments were performed in Ringer and Ca²⁺-free Ringer solutions. Confocal Ca²⁺ imaging was performed using an Olympus Fluoview confocal scanning system fitted to IX70 microscope using a ×10 (0.3 numerical aperture) objective. Images (256 × 256 pixels) were collected and analyzed using Olympus Fluoview software.

CaMKII Assay

CaMKII kinase activity was measured by lysing oocytes (20 μl/oocyte) in CaMKII extraction buffer (80 mm β-glycerophosphate, 20 mm Hepes, pH 7.5, 15 mm MgCl₂, 1 mm sodium vanadate, 50 mm NaF, 1 mm dithiothreitol, 1× protease inhibitor mixture (Calbiochem)). Lysates were centrifuged at 500 × g for 15 min, and the supernatant was stored at − 70 °C until use in the kinase assay. The CaMKII kinase was performed using the SignaTECT^TM kinase kit (Promega) according to manufacturer’s instructions.

RESULTS

Intracellular Ca²⁺ Potentiates SOCE

Physiological activation of SOCE is invariably preceded by a rise in Ca²⁺_i because of Ca²⁺ release from stores. Although it is clear that Ca²⁺_i is not required for SOCE activation, it is not known whether this Ca²⁺_i modulates I_SOC. To address this issue, we activated SOCE under conditions that either allow an intracellular Ca²⁺ rise or not. To activate SOCE in the absence of a Ca²⁺_i rise, we injected cells with BAPTA and then depleted Ca²⁺ stores with ionomycin (BAPTA-Ion). This results in the activation of a characteristic SOCE current as previously described (Fig. 1B, filled squares) (14, 17). To allow a Ca²⁺_i rise during SOCE activation, Ca²⁺ stores were depleted with ionomycin followed by repetitive hyperpolarization to induce Ca²⁺ influx (Ion- BAPTA). The cell was then injected with BAPTA, and I_SOC was recorded (Fig. 1B, open circles). Treating cells according to the Ion-BAPTA protocol resulted in a significant (p = 1.6 × 10⁻⁷) potentiation of I_SOC levels compared with control oocyte (BAPTA-Ion) (Fig. 1, B and C). Therefore, allowing a Ca²⁺_i rise during SOCE activation leads to current potentiation.

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).

The Ca²⁺-mediated potentiation of SOCE described above was obtained under conditions designed to maximize Ca²⁺_i rise by hyperpolarization-induced Ca²⁺ influx. To determine whether such Ca²⁺ influx is required for I_SOC potentiation, a similar experiment was performed without hyperpolarization using the ion-BAPTA protocol, that is Ca²⁺ stores were simply depleted in the absence of BAPTA injection. In this case, I_SOC was also potentiated (Fig. 1D), showing that hyperpolarization per se is not required for the observed Ca²⁺_i-mediated I_SOC potentiation.

As discussed above, SOCE has been shown to be potentiated by extracellular Ca²⁺, which affects SOCE channel gating in a process referred to as CDP (12). To avoid confusion with CDP, we will refer to the intracellular Ca²⁺ effect on I_SOC as Ca²⁺_i-mediated potentiation (CMP). CMP provides a fitting feedback mechanism between Ca²⁺ release and SOCE. This is especially relevant because as shown by Parekh et al. (18), SOCE activation is an all or nothing process (18). That is even partial depletion of intracellular Ca²⁺ stores results in full activation of I^SOC. To determine the relationship between store Ca²⁺ load and I_SOC magnitude in Xenopus oocytes, SOCE was measured by Ca²⁺ imaging in voltage-clamped cells, which allows simultaneous recording of Ca²⁺_i and SOCE in the same cell (Fig. 2) (19). Membrane potential was stepped repetitively to +40 mV (where Ca²⁺ influx is minimal) and −140 mV (to induce Ca²⁺ influx through SOCE). Under these conditions, the difference in Ca²⁺ fluorescence at −140 and +40 mV (Fig. 2, open triangles) provides a reliable measure of SOCE and correlates well with I_SOC (19). To induce partial store depletion, cells were treated with thapsigargin (1 μm) for 1 h (complete store depletion requires ≥3 h), which results in SOCE activation as indicated by the increased fluorescence at −40 V (Fig. 2, open circles). Injection of IP₃ leads to further Ca²⁺ release (Fig. 2, squares & circles), confirming that thapsigargin induced partial store depletion. However, the additional store depletion following IP₃ injection does not augment SOCE (Fig. 2, open triangles), indicating that SOCE magnitude does not correlate well with the extent of store depletion. This shows that increased store depletion, at least in the range tested, does not translate into a larger SOCE, arguing that SOCE is modulated by other signaling pathways after store depletion. Fig. 1 shows that one such modulatory pathway is the Ca²⁺_i levels during SOCE activation.

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.

Correlation between Receptor-induced Ca²⁺ Mobilization and SOCE

To assess the physiological relevance of CMP, we sought to determine whether it could be observed following IP₃-linked receptor stimulation. We were interested in inducing different levels of Ca²⁺_i rise while minimizing experimental manipulation of Ca²⁺_i (such as BAPTA injection or ionomycin treatment). Our approach was to express the G-protein-coupled serotonin receptor (5HT1c) and monitor changes in the endogenous Ca²⁺-activated Cl⁻ currents as markers of Ca²⁺_i (19). 5HT1c stimulation with serotonin (5HT) results in IP₃ production through phospholipase-β activation (20). We have previously shown that Ca²⁺-activated Cl⁻ currents (I_Cl1 and I_Cl1T) faithfully report Ca²⁺_i changes below the plasma membrane in terms of amplitude and kinetics (19). I_Cl1 activates in response to Ca²⁺ release from internal stores, whereas I_Cl1T responds to Ca²⁺ influx from the extracellular space. During Ca²⁺ release, I_Cl1 activates as a sustained outward current upon depolarization (+ 40 mV, Fig. 3A, upper trace). Ca²⁺ release results in store depletion and SOCE activation. Ca²⁺ flowing through SOCE channels activates I_Cl1T as a transient current, only when the depolarization pulse is preceded by a hyperpolarization step, which induces Ca²⁺ influx (Fig. 3A, lower trace). I_Cl1T is transient because the Ca²⁺ that enters through SOCE channels during the −140-mV pulse dissipates rapidly during the subsequent 40-mV pulse, leading to I_Cl1T current decay (Fig, 3A, lower trace) (19) (for a more detailed description of the relationship between Ca²⁺ signals and Ca²⁺-activated Cl⁻ currents, see Refs. 19, 21, and 22). Thus, monitoring I_Cl1 and I_Cl1T allows the real-time determination of Ca²⁺_i (I_Cl1) and SOCE (I_Cl1T) levels following 5HT1c activation.

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).

To generate graded Ca²⁺ release responses and determine the effect on SOCE, we injected cells with different amounts of 5HT1c cRNA (10 or 30 ng) and allowed them to express for 2 days. Cells were then stimulated with 5HT (10 μm) (Fig. 3, B and C), leading to Ca²⁺ release, which activates I_Cl1 (Fig. 3, B and C, squares) followed by I_Cl1T due to SOCE activation (Fig. 3, B and C, circles). I_Cl1T activates to a certain threshold and gradually returns to base line as a result of Ca²⁺ store refilling and SOCE inactivation (Fig. 3, B and C, circles). Cells injected with 10 ng of 5HT1c cRNA produced a smaller I_Cl1 (Fig. 3B) than those injected with 30 ng of the receptor (Fig. 3C). Furthermore, I_Cl1T activated to a lower threshold and inactivated more rapidly in cells injected with 10 ng of 5HT1c receptor cRNA (Fig. 3, compare B with C, circles). The responses of the Cl⁻ currents summarized in Fig. 3D show that both Ca²⁺_i (as indicated by I_Cl1) and SOCE (as indicated by I_Cl1T) were significantly enhanced at high 5HT1c (30 ng) expression. Therefore, enhanced Ca²⁺ release correlates with larger SOCE. A simple explanation for these data is that receptor stimulation in cells expressing high levels of 5HT1c leads to a more dramatic Ca²⁺ store depletion and larger SOCE. However, as shown in Fig. 2, the extent of Ca²⁺ store depletion does not linearly correlate with SOCE magnitude. Therefore, SOCE potentiation as reported by I_Cl1T in cells expressing more 5HT1c receptors is probably because of some mechanism other than the extent of store depletion. CMP of I_SOC provides such a mechanism. High 5HT1c expression produces increased Ca²⁺_i as indicated by I_Cl1, which would be expected to potentiate I_SOC (I_Cl1T) through CMP. Note that store depletion provides a required signal for SOCE activation, but the extent of store depletion per se does not modulate SOCE magnitude. Rather, the data in Figs. 2 and 3 argue that it is the magnitude of the Ca²⁺_i rise that modulates SOCE through CMP. Clearly the magnitude of Ca²⁺_i rise can correlate with the extent of store depletion. These data reveal a subtle but important distinction in the mechanism of SOCE modulation and are consistent with the notion that CMP is a physiologically relevant modulator of SOCE.

The positive correlation between Ca²⁺_i and SOCE described above is not limited to Xenopus oocytes. Gailly et al. (23) described a similar relationship in Chinese hamster ovary cells (23), arguing that CMP is a widespread mechanism of SOCE regulation.

CaMKII Potentiates SOCE

Ca²⁺_i-mediated potentiation of I_SOC could be either direct or indirect through the activation of Ca²⁺-dependent downstream effectors. A direct Ca²⁺-mediated potentiation of I_SOC is unlikely because Ca²⁺ has been shown to inactivate I_SOC, probably through a direct effect on SOCE channels (fast Ca²⁺-dependent inactivation) (8, 10). This finding suggests that CMP is the result of activation of Ca²⁺-dependent effectors, which in turn act on SOCE. A primary candidate for such an effector pathway is the Ca²⁺-CaM-activated protein kinase pathway. The most widespread Ca²⁺-CaM-dependent kinase is CaMKII, which is expressed in Xenopus oocytes (24). If Ca²⁺ potentiates I_SOC through CaMKII, it is expected that ectopic activation of CaMKII would lead to increased I_SOC levels.

We used a constitutively active CaMKII mutant (CaMKII^ca) to determine whether CaMKII activation potentiates I_SOC. The CaMKII holoenzyme is a multisubunit complex that is kept inactive by an autoinhibitory domain. The binding of Ca²⁺- CaM relieves autoinhibition and stimulates autophosphorylation at Thr-286, rendering the enzyme Ca²⁺-CaM-independent (25). CaMKII^ca is a T286D mutation that mimics the effects of autophosphorylation by replacing Thr-286 with Asp, resulting in a Ca²⁺-CaM-independent and thus constitutively active CaMKII (26). We injected oocytes with CaMKII^ca and measured I_SOC (Fig. 4A). Expression of CaMKII^ca results in an ~3-fold increase in I_SOC (p = 1.1 × 10⁻⁷) (Fig. 4, A and B), consistent with CMP acting through CaMKII. In addition, CaMKII^ca-mediated I_SOC potentiation is expected to be independent of Ca²⁺_i because CaMKII^ca is Ca²⁺-CaM-independent. This prediction is confirmed in CaMKII^ca-expressing cells, as I_SOC is potentiated whether store depletion is preceded by a rise in Ca²⁺_i (Ion-BAPTA) or not (BAPTA-Ion) (Fig. 4C). This finding further suggests that Ca²⁺-mediated potentiation is primarily through CaMKII activation, because no additive effect on I_SOC is observed in CaMKII^ca-expressing cells subjected to the Ion-BAPTA protocol (Fig. 4C).

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).

To directly confirm CaMKII^ca expression, we measured CaMKII-specific activity in lysates from control and CaMKII^ca-injected cells. Cells expressing CaMKII^ca had higher levels of CaMKII activity (Fig. 4D), showing that CaMKII^ca was expressed and functional in these cells. Because specific activity was measured in the absence of Ca²⁺-CaM (Fig. 4D), CaMKII activity data confirm the Ca²⁺-CaM independence of CaMKII^ca.

It is important to note that the expression of CaMKII^ca by itself is not sufficient to activate I_SOC (Fig. 4A, circles). In CaMKII^ca-expressing cells, store depletion is still required to activate SOCE because no I_SOC is detected before ionomycin treatment (Fig. 4A, circles). However, I_SOC levels reach a significantly larger maximal amplitude in CaMKII^ca-expressing cells compared with control cells (Fig. 4, A and B). Therefore, CaMKII is not a component of the SOCE activation pathway induced in response to store depletion but rather positively modulates SOCE activity following SOCE activation.

If CMP is acting through CaMKII, CaMKII should be activated in cells where a Ca²⁺_i rise is induced (Ion-BAPTA). We were unable to detect such an increase in CaMKII activity in oocytes treated according to the Ion-BAPTA protocol. This result argues that either Ca²⁺ and CaMKII potentiate I_SOC by separate mechanisms or that CaMKII activation is transient and/or spatially localized, resulting in small changes in CaMKII activity that are difficult to detect in whole cell lysates. Furthermore, basal CaMKII activity was quite variable in oocytes donated from different females, making it difficult to reliably measure a small increase in CaMKII activity in different batches of cells. However, a Ca²⁺_i rise has been shown to activate endogenous CaMKII in Xenopus oocytes using an in vivo CaMKII-specific kinase assay (27).

Nonetheless, if CMP is mediated by CaMKII, blocking endogenous CaMKII should inhibit CMP. Therefore, we blocked endogenous CaMKII and determined the effect on SOCE. Oocytes were injected with AIP, a specific inhibitory peptide of CaMKII that mimics the autoinhibitory domain and blocks CaMKII activity (28). Allowing a Ca²⁺_i rise in control oocytes (Ion-BAPTA) potentiates I_SOC by ~2-fold but not in cells injected with the CaMKII inhibitor AIP (Fig. 5A). Furthermore, AIP added to the CaMKII assay blocks kinase activity in a dose-dependent manner (Fig. 5B). These data show that inhibiting endogenous CaMKII activity blocks CMP, supporting the conclusion that Ca²⁺_i potentiates SOCE through CaMKII activation.

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).

Mechanism of CaMKII Action on SOCE

Because SOCE is activated in response to store depletion, CaMKII could potentiate I_SOC by targeting either the coupling mechanism between Ca²⁺ stores and SOCE or the SOCE channel. To differentiate between these possibilities and obtain a better understanding of the mechanism of action of CaMKII, we wanted to study the effects of CaMKII independently of store depletion. This was accomplished by irreversibly depleting Ca²⁺ stores with thapsigargin before CaMKII^ca expression (Fig. 6). Oocytes were incubated in thapsigargin (1 μm) for 3 h to fully deplete Ca²⁺ store followed by CaMKII^ca cRNA injection and incubation in nominally Ca²⁺-free (50 μm free Ca²⁺) solution. Control cells were treated with thapsigargin alone. Under these conditions, store depletion is complete before CaMKII^ca expression and store Ca²⁺ load remains low throughout the experiment because thapsigargin irreversibly inhibits the ER Ca²⁺-ATPase. This allows us to study the effects of CaMKII on I_SOC after store depletion and determine whether CaMKII is affecting the coupling mechanism or SOCE channel gating or permeation. In this experiment, SOCE was activated in the absence of the conducting ion (Ca²⁺) and therefore no SOCE current was observed at the beginning of the experiment (Fig. 6A). Switching control (thapsigargin-treated) oocytes to a Ca²⁺-containing solution produced an initial I_SOC that was further enhanced over time, eventually saturating (Max I_SOC) within ~6 min (Fig. 6A, squares). This behavior is the result of classical CDP where extracellular Ca²⁺ exerts a positive effect on SOCE channel gating (12, 13). CDP can be estimated as the ratio of maximal I_SOC/initial I_SOC (12) and is 3.11 ± 0.23 in thapsigargin-treated cells (Fig. 6B).

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).

Surprisingly, exposing CaMKII^ca-expressing cells to Ca²⁺- containing solution results in an initial I_SOC with a similar amplitude to initial I_SOC in control cells (Fig. 6A, open circles). However, I_SOC in CaMKII^ca-expressing cells gradually increased to significantly higher levels than in control cells (Fig. 6A, open circles). This shows that CaMKII^ca expression does not affect initial I_SOC levels but results in a significantly (p = 1.46 × 10⁻⁵) larger maximal I_SOC (Fig. 6B). It follows that CDP, calculated as the ratio of maximal/initial I_SOC, was also augmented (5.72 ± 0.61) in CaMKII^ca-expressing cells (Fig. 6B). Therefore, CaMKII potentiates I_SOC by increasing the levels of CDP without affecting initial I_SOC levels, even after prolonged expression of CaMKII^ca.

These data provide important insights into the mechanism of action of CaMKII. The whole cell SOCE current is defined by the following equation: I_SOC = NP_oi, where N is the number of active channels, P_o is the probability of opening, and i is the single channel conductance. Therefore, CaMKII can potentiate I_SOC by increasing either N, i, or P_o. If CaMKII was affecting the coupling mechanism, an increase in the number of active channels (N) is expected. The fact that CaMKII^ca expression does not enhance initial I_SOC provides evidence against an increase in channel number (N) or single channel conductance (i) (Fig. 6, A and B). This is because initial I_SOC is due to current flowing through all of the open channels after Ca²⁺ addition. If either N or i was augmented by CaMKII, initial I_SOC would be larger in CaMKII^ca-expressing cells. This hypothesis argues that CaMKII potentiates I_SOC by enhancing the probability of opening (P_o) of SOCE channels.

The observed increase in CDP levels in CaMKII^ca-expressing cells strongly supports the conclusion that CaMKII potentiates I_SOC by increasing P_o. This is because CDP has been shown to be the result of extracellular Ca²⁺ exerting a positive effect on SOCE channel gating (P_o) (12, 13).

Representative current traces in response to a step pulse to −120 mV (Fig. 6C) and a voltage ramp from −140 to 50 mV (Fig. 6D) obtained from control (Thaps) and CaMKII^ca-injected cells (Thaps-CaMK^ca) are shown. The time points during the experiments at which the traces were obtained are indicated in Fig. 6A (Thaps, open symbols; Thaps-CaMK^ca, filled symbols). Note the similar levels of initial I_SOC in both treatments (Fig. 6C, open and filled stars). Initial I_SOC traces from CaMKII^ca-injected cells show a time-dependent increase in current amplitude (Fig. 6C, filled star) that is due to CDP occurring during the 500-ms duration of the voltage pulse. Zweifach and Lewis (8) have shown that the extent of Ca²⁺-dependent inactivation of I_CRAC following a brief hyperpolarization depends on the single channel current (i) (8). That is, increased unitary current (i) results in a faster rate of I_CRAC inactivation. Assuming that the same relationship holds for Xenopus oocyte I_SOC, if CaMKII increases unitary conductance (i), we expect a faster I_SOC inactivation rate shortly after the hyperpolarization pulse. However, the rate of I_SOC inactivation shortly after (100 ms) hyperpolarization is similar between control and CaMKII^ca-expressing cells (Fig. 6E). In fact, at steady-state, control cells exhibit a more marked I_SOC inactivation (Fig. 6E). These inactivation kinetics argue that CaMKII does not enhance I_SOC unitary conductance. Superimposed current-voltage relationships from control and CaMKII^ca-injected cells were similar (Fig. 6F), indicating that CaMKII does not affect the voltage dependence of I_SOC.

These results argue that CaMKII^ca potentiates I_SOC by targeting SOCE channel gating and not the coupling mechanism between Ca²⁺ stores and SOCE. Three pieces of evidences support the conclusion that CaMKII potentiates I_SOC by affecting channel gating (P_o) and not channel number (N) or unitary conductance (i): 1) similar levels of initial I_SOC in control and CaMKII^ca-expressing cells (Fig. 6, A and B) arguing against an effect of CaMKII on N or i. 2) enhanced CDP in CaMKII^ca-expressing cells (Fig. 6B), suggesting that CaMKII enhances channel P_o. 3) similar I_SOC inactivation rates shortly after hyperpolarization in control and CaMKII^ca-expressing cells (Fig. 6E) arguing against an increase in unitary conductance (i).

DISCUSSION

Ca²⁺_i has been shown to negatively regulate I_SOC by inducing channel inactivation either directly or through store refilling (8, 10, 11). Here we show that Ca²⁺_i can in addition have a potentiating effect on SOCE. The Ca²⁺_i effect on SOCE is probably the result of CaMKII activation, because Ca²⁺_i, because inhibition of endogenous CaMKII activity blocks CMP and expression of CaMKII^ca is sufficient to potentiate I_SOC independently of Ca²⁺_i. Although CaMKII potentiates I_SOC, it is not sufficient by itself to activate SOCE independently of store depletion (Fig. 4A). This observation is consistent with the fact that a Ca²⁺_i rise is not required for SOCE activation (1). Therefore, the CaMKII pathway modulates SOCE activity but is not an essential component of the SOCE activation pathway in response to store depletion. For CaMKII to exert its effects, SOCE has to be activated by store depletion.

Using pharmacological inhibitors, a role for CaMKII in skeletal muscle SOCE (29) and myosin light chain kinase (a Ca²⁺- CaM-dependent kinase related to CaMKII (25)) in endothelial SOCE (30) have been postulated. This finding argues that CaMKII modulation of I_SOC is a widespread mechanism that is not cell type-specific. Nonetheless, a previous report by Matifat et al. (27) suggests a negative effect of CaMKII on SOCE in Xenopus oocytes. However, it is not clear from this study that the reported CaMKII effect was due to SOCE modulation because the Ca²⁺-activated Cl⁻ current was used as an indicator of SOCE, making it impossible to differentiate between an effect of CaMKII on the Cl⁻ current or SOCE. In contrast, we have directly measured the SOCE current while modulating CaMKII activity and show that CaMKII activation potentiates I_SOC. This finding argues that the effects of CaMKII reported by Matifat et al. (27) are due to modulation of the Cl⁻ currents or other Ca²⁺ influx pathways in the oocyte.

CaMKII provides an excellent modulator of SOCE activity because of its ability to decode spatial and temporal information encoded in Ca²⁺_i signals into different levels of kinase activity. This capacity is attributable to spatial and structural features of CaMKII. CaMKII localizes to specific subcellular compartments such as the nucleus and cytoskeleton (31, 32), and its activity increases exponentially based on the number and frequency of Ca²⁺_i oscillation (15, 33). These exceptional attributes allow CaMKII to provide specificity to Ca²⁺_i signals by differentially activating effectors based on the kinetics of Ca²⁺_i signals. CaMKII plays important roles in regulating various cellular functions such as gene expression, cell cycle progression, and learning and memory (25, 34). The data presented here show that SOCE can now be added to the list of cellular functions modulated by CaMKII.

In a similar fashion to the CaMKII effect on SOCE described here, CaMKII has been shown to augment both L-type (Ca_v1.x) (35) and T-type (Ca_v3.x) (36) Ca²⁺ currents. CaMKII regulation of SOCE described in this paper is reminiscent of Ca²⁺-dependent facilitation of inward L-type Ca²⁺ current. As Ca²⁺_i increases, L-type Ca²⁺ current is enhanced in a process termed facilitation (37). This facilitation is mediated by CaMKII, which increases the open probability of L-type channels (35). Our data strongly argue that CaMKII potentiates I_SOC in a similar fashion by altering channel gating (see Fig. 6). This similarity in the mechanism of action of CaMKII on both voltage- gated and store-operated Ca²⁺ channels argues for a common cross-talk between Ca²⁺_i kinetics and Ca²⁺ influx pathways. It is attractive to postulate that CaMKII provides a mechanism for phospholipase-linked receptors to differentially modulate SOCE activity. That is, the spatiotemporal features of the Ca²⁺_i signals downstream of receptor activation differentially activate CaMKII and thus SOCE.

In addition to the CaMKII-mediated regulation of SOCE described here, SOCE has been shown to be modulated by protein kinase C (38, 39). Both protein kinase C and CaMKII are downstream kinases that can be induced following activation of phospholipase-linked receptors. Therefore, the interplay between various modulatory mechanisms in a specific cellular context combines to generate different levels of Ca²⁺ influx through SOCE. This Ca²⁺ influx affects Ca²⁺_i kinetics and thus Ca²⁺-dependent cellular responses. Consequently, CaMKII regulation of SOCE is likely to have broad and important physiological consequences.