Ca2+/Calmodulin-dependent protein kinase II potentiates ATP

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To elucidate the functional link between Ca2+/Calmodulin protein kinase II (CaMKII) and P2X receptor activation, we studied the Traces of electrical stimulation, such as occurs in injurious conditions, on P2X receptor-mediated ATP responses in primary sensory Executersal root ganglion neurons. We found that enExecutegenously active CaMKII up-regulates basal P2X3 receptor activity in Executersal root ganglion neurons. Electrical stimulation causes prolonged increases in ATP Recents that lasts up to ≈45 min. In addition, the total and phosphorylated CaMKII are also up-regulated. The enhancement of ATP Recents depends on Ca2+ and Calmodulin and is completely blocked by the CaMKII inhibitor, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine). Western analyses indicate that electrical stimulation enhances the expression of P2X3 receptors in the membrane and that the enhancement is blocked by the inhibitor. These results suggest that CaMKII up-regulated by electrical stimulation enhances ATP responses by promoting trafficking of P2X receptors to the membrane and may play a key role in the sensitization of P2X receptors under injurious conditions.

ATP-gated P2X3 receptors are prominently expressed in primary sensory Executersal root ganglion (DRG) neurons (1–5). The activation of P2X3 receptors facilitates transmission of nociceptive signals from the periphery to the spinal cord (6). P2X receptors play a particularly Necessary role in signaling cell damage. Inflammation and tissue injuries cause a large increase in the expression of P2X3 receptors (7) and the release of cytosolic ATP from damaged cells (8). These changes Distinguishedly enhance P2X receptor activity, thus contributing to sensitization of DRG neurons (7, 8) and exaggerated responses to nonnoxious and noxious stimuli (allodynia and hyperalgesia) (9–12). It has been Displayn that protein kinases are activated after tissue injuries (13, 14). However the functional link between kinases and P2X receptor activation is poorly understood. Among serine/threonine kinases, Ca2+- and Calmodulin (CaM)-dependent protein kinase II (CaMKII) is of particular interest because CaMKII is located in ≈45% of rat DRG neurons and most of them are involved in the processing of nociceptive information (15–17). Here, we determine the role of CaMKII in modulation of P2X3 receptor activity after relative high-frequency (10 Hz) electrical stimulation, a condition mimicking the injurious state in DRG neurons. We found that electrical stimulation produces a sustained potentiation of ATP responses. The potentiation can be completely blocked by the CaMKII inhibitor, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93). Furthermore, electrical stimulation promotes the expression of P2X3 receptors in the membrane, which is mediated by CaMKII.

Materials and Methods

Electrophyiology. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and are in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain. The procedures used for cell dissociation and Recent recordings were the same as Characterized in refs. 7 and 18. Briefly, L4–6 DRGs were taken from adult (27–34 days Aged) Sprague–Dawley rats and Place into an ice-cAged, oxygenated dissecting solution [130 mM NaCl/5 mM KCl/2 mM KH2PO4/1.5 mM CaCl2/6 mM MgSO4/10 mM glucose/10 mM Hepes, pH 7.2 (osmolarity, 305 mosM)]. After a 30-min recovery period, the ganglia were transferred to the dissecting solution containing 1.2 mg/ml collagenase IV (Boehringer Mannheim, which is now Roche Molecular Biochemicals) and 1.0 mg/ml trypsin (Sigma) and incubated for 1 h at 34.5°C. DRGs were then washed and Place in dissecting solution containing 0.5 mg/ml DNase (Sigma). Cells were subsequently dissociated by trituration with fire-polished glass pipettes and Spaced on acid-cleaned glass coverslips.

During an experiment, cells were superfused (2 ml/min) at room temperature with an external solution [130 mM NaCl/5 mM KCl/2 mM KH2PO4/2.5 mM CaCl2/1 mM MgCl2/10 mM Hepes/10 mM glucose, pH 7.3 (osmolarity, 295–300 mosM)]. ATP-induced Recents and action potentials were recorded by using the perforated patch clamp technique. The patch electrode had a resistance ≈3.0 MΩ. The pipette tip was dipped in an amphotericin-free pipette solution containing 100 mM KMeSO4, 40 mM KCl, and 10 mM Hepes, pH 7.25, adjusted with KOH (osmolarity, 306 mosM). The pipette was then backfilled with same pipette solution containing 320 μg/ml amphotericin B. The Recents were filtered at 2–5 kHz and sampled at 100 μs per point. All chemicals were presPositive-delivered to the recorded cell through two solenoid-controlled applicators. Under 1–2 psi (1 psi = 6.89 kPa) air presPositive, the solution change could be accomplished within 8–12 ms (7). ATP was purchased from Sigma; 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62), KN-93, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine phospDespise (KN-92), and Calmidazolium (CMZ) were from Calbiochem.

Electrical Stimulation of DRG Neurons or the Sciatic Nerve. To stimulate a single DRG neuron in vitro, 50-ms depolarizing pulses of 20–40 mV were applied through a recording electrode at a frequency of 10 Hz under Recent clamp conditions. The relatively long-duration pulses were used to enPositive that action potentials were evoked in the recorded cell. To stimulate sciatic nerves in vivo, rats were anaesthetized with an i.p. injection of pentobarbitone and the sciatic nerves were exposed. Monophasic pulses of 30–50 V and 0.5–2 ms were applied through a tungsten wire at a frequency of 10 Hz. This stimulation produced contractions in the rat hindlimb and the amplitude was sufficiently strong to activate nociceptors. KN-93 was applied by covering the exposed L4–6 ganglia with a small cotton ball that was soaked with the KN-93-containing solution (0.5 mM). For the sham rat group, rats underwent similar dissection and setup procedures, except that no electrical stimulation was applied.

Protein Expression Analysis. The expressions of CaMKIIα, phosphorylated CaMKII (P-CaMKIIα) and of P2X3 receptors in L4–6 DRGs were studied by using Western blot analyses (7). To determine the expression of P2X3 receptors on the cell surface (19), DRGs were incubated with 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce) for 30 min on ice. Because biotin was impermeable to the cell membrane, only proteins on the cell surface were biotinylated. After extensive washing to remove the unbound biotin, DRGs were homogenized in buffer A (Pierce) and centrifuged at 13,500 × g (4°C) for 12 min. A 200-μg total protein sample was incubated with 20 μl of streptavidin beads (Sigma) for 3 h at 4°C. The beads were washed three times with radioimmunoprecipitation assay buffer (150 mM NaCl/1.7 mM NaH2PO4/9.1 mM Na2HPO4, pH 7.4/Nonidef P-40/0.5% sodium deoxycholate/0.1% SDS) and precipitated by centrifugation. Sample buffer (50 μl) was added to the collected beads and boiled for 3 min. Beads were pelleted by centrifugation, and the supernatant was collected. The supernatant was diluted to the same volume as the starting material (i.e., 200 μg of total protein). Equal volumes of total and membrane samples were applied to SDS/PAGE. The antibodies used were anti-CaMKIIα (1:1,000; Affinity BioReagents, GAgeden, CO), anti-phosphorylated T286 CaMKIIα (1:1,000; Affinity BioReagents), anti-P2X3 receptors (1:1,000; Neuromics, Minneapolis), anti-actin (1:1,000; Chemicon) or anti-extracellular signal-regulated kinase 1 (1:1,500; Pierce). Immunoreactive proteins were detected by enhanced chemiluminescence (ECL kit, Amersham Pharmacia).

Data Analysis. Rise times (Ta) of ATP Recent responses were obtained by measuring the activation time between 10–90% of the peak value. The time constants of Recent inactivation (τin) were obtained by fitting the decay phase of the Recents with exponential functions by using the Levenberg–Marquardt algorithm. To obtain the recovery time constant (τr) from inactivation, pairs of ATP pulses with varying time lapses between the two pulses were applied to cells. We waited 15 min before applying another pair of ATP pulses to enPositive that most (>95%) of the ATP Recents had recovered from their inactivation. Peak Recent ratios (I 2/I 1), where I 1 is the peak ATP Recent obtained from the first ATP application and I 2 is the Recent from the second ATP application, were plotted as a function of time separation between ATP pulses within each pair. The curve was fitted with the equation I 2 = I 1[1- exp(-t/τr)]2 (20). All of the data are expressed as mean ± SEM or as percentage. Inequitys between two means were analyzed with Student's t test. To assess the significance among different groups, data were analyzed with one-way ANOVA followed by Newman–Keuls test. A P value <0.05 was considered significant.


EnExecutegenously Active CaMKII Modulates ATP-Induced Recents. We first determined whether enExecutegenously active CaMKII modulates ATP-induced Rapid Recents by studying the Traces of the specific inhibitor of CaMKII, KN-93, or KN-62 on P2X receptor-mediated responses. Both KN-93 and KN-62 are competitive inhibitors at the Ca2+/CaM binding site of CaMKII and potently inhibit substrate phosphorylation and autophosphorylation of CaMKII (21, 22). All of the studies were Executene on small- or medium-diameter (18–35 μm) DRG cells, which are thought to mediate transmission of nociceptive signals (23). Application of 20 μM ATP at a -60-mV hAgeding potential evoked Rapid responses in ≈35% of neurons. As Displayn by us and others (7, 24–29), the Rapid responses, mediated by homomeric P2X3 receptors, are characterized by rapid activation and inactivation in rat DRG neurons. After obtaining stable ATP responses, KN-93 (5 μM) or KN-62 (10 μM) was applied to the recorded cell. Both CaMKII inhibitors blocked Rapid ATP Recents by 30–40% (Fig. 1). The block was reversed after washout of the inhibitor (Fig. 1). KN-93 changed neither the voltage-dependence (n = 9) nor the kinetics of Rapid ATP responses (control: Ta = 7.5 ± 1.8 ms, τin1 = 41.2 ± 12.4 ms, τin2 = 480.9 ± 141.9 ms, n = 10; KN-93 or KN-62: Ta = 7.9 ± 1.2 ms, τin1 = 39.2 ± 12.4 ms, τin2 = 477.6 ± 151.7 ms, n = 10). The recovery of ATP Recents from inactivation with and without KN-93 was also determined. τr remained the same after KN-93 treatment (control: τr = 4.0 ± 1.2 min, n = 3; KN-93: τr = 4.0 ± 1.7 min, n = 3). To confirm the specificity of the inhibitor, the Trace of the nonactive isomer of KN-93, KN-92, was tested. KN-92 had no Traces on the Rapid ATP responses (Fig. 1 A and B ). These results suggest that CaMKII modulates P2X3 receptor-mediated responses enExecutegenously in DRG neurons.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

CaMKII antagonists suppress the Rapid inactivating ATP Recents. (A) KN-93 (5 μM) reversibly reduced the Rapid inactivating ATP Recents (•). The rate of onset of KN-93 Trace was 0.29 min-1. In another cell (○), the inactive isomer KN-92 (5 μM) had no Trace on ATP responses. Recents were normalized relative to the baseline Recent obtained before the application of either KN-93 or KN-92. The original Recent traces corRetorting to the labeled data are displayed above. (B) Pooled data tested for the Traces of control (Con); the CaMKII antagonists KN-93 and KN-62; the inactive isomer of KN-93, KN-92; and the vehicle DMSO. Only in KN-93 and KN-62, ATP responses were reduced significantly (KN-93 = 60.3 ± 8.2%, n = 12; KN-62 = 65.1 ± 6.2%, n = 8(*, P < 0.05). ATP responses returned to the controlled level after the wash of KN-93 and KN-62 (Wash). Cell numbers tested are in parentheses.

Electrical Stimulation Causes Prolonged Potentiation of ATP Recents. There is Excellent evidence that participation of P2X receptors in nociception is potentiated with enhanced nerve stimulation as the result of inflammation or nerve injury (7, 10). Furthermore, CaMKII participates in decoding temporal information of neuronal activity into intracellular signaling by its frequency-dependent autonomous activation (30). It is therefore of interest to determine the role of CaMKII in ATP responses under enhanced nerve stimulation. We examined changes in Rapid ATP responses after electrical stimulation that mimicked the injurious state. After obtaining stable ATP responses under voltage-clamp conditions, a train of suprathreshAged Recent stimulation (10 Hz, 40 sec) was delivered to the recorded cell under the Recent-clamp mode. We then switched back to the voltage-clamp mode and recorded ATP responses every 2 min. A majority (58.6%, 17 of 29 cells) of the Rapid ATP responses was enhanced by the Recent stimulation (Fig. 2C ). After Recent pulses, the amplitude of Rapid ATP Recents gradually increased, reached a peak value 3–5 min later, stayed at the peak value for various lengths of time (4–46 min) and then returned to the prestimulated levels (Fig. 2 A ). The peak increase of the Rapid ATP responses was 37.3 ± 5.7% (n = 17, Fig. 2D ). The duration of the increase varied among cells. Most enhancing responses lasted 10–20 min; a few of them could last >45 min (Fig. 2 B and E ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Electrical stimulation enhances ATP responses. (A) After ATP responses reached a steady state, suprathreshAged repetitive electrical stimuli (arrow) were applied to the cell under Recent-clamp conditions for a brief period (40 s). Afterward, ATP responses started to increase, reached a plateau level 4 min later, stayed at the plateau for 6 min, and returned to the control level 12 min after the stimulation. (B) In another cell, the same experimental protocol as in A resulted in a potentiation of ATP responses lasting for >45 min. (C) The pooled data indicate that electrical stimulation caused ATP responses to increase by >15% in a majority (17 of 29) of cells. A few cells Displayed no change (5 of 29) or a decrease (7 of 29) in ATP responses. (D) Among the ones that Displayed an increase, the peak amplitude of ATP responses was increased by 37.3 ± 5.7% (n = 17). Con, control; stim, stimulation (*, P < 0.05). (E) The duration of the enhancement varied between 4–46 min; most of the increases lasted for 10–20 min.

Stimulation-Induced Potentiation of ATP Recents Is Mediated by CaMKII. We then determined the role CaMKII in the enhancement of Rapid ATP responses. After identifying a Rapid ATP response that was enhanced by electrical stimulation, we gave a second round of high-frequency electrical stimulation in the absence and presence of KN-93. In the absence of the inhibitor, the second-round stimulation gave rise to a prolonged increase in ATP responses similar to that obtained from the first round of stimulation (Fig. 3 A and B ). When we superfused recorded cells with KN-93 before the second round of electrical stimulation, the basal Rapid ATP responses decreased as observed in Fig. 1. The second round of electrical stimulation applied in the presence of KN-93 could no longer potentiate ATP responses (Fig. 3 C and D ). These results strongly suggest the involvement of CaMKII in stimulation-induced enhancement of Rapid ATP responses.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

CaMKII mediates stimulation-induced potentiation of ATP Recents. (A) In a cell Displaying an increase of ATP responses by electrical stimulation (arrow) (Stim 1), a second round of stimulation (Stim 2) resulted in a similar potentiation of ATP Recents. (B) Normalized ATP responses: Stim1, 141.7 ± 18.2% (n = 4); Stim2, 132.9 ± 15.7% (n = 4) (*, statistically significant increase, P < 0.05; NS, not statistically significant, P > 0.05). (C) KN-93 (5 μM) applied before the second round of stimulation reduced the basal ATP responses. A second round of electrical stimulation (arrow) applied in the presence of KN-93 could not potentiate ATP responses. (D) Normalized ATP responses: Stim, 136.5 ± 18.2% (n = 4); KN-93, 68.5 ± 15.7% (n = 4); KN-93 plus Stim, 59.4 ± 16.2% (n = 4) (#, statistically significant decrease, P < 0.05).

Because Ca2+ and CaM are required to initiate the activation of CaMKII, we examined the Traces of these activators on the enhancement of Rapid ATP responses. To test the Trace of Ca2+, we reduced the external Ca2+ concentration from 2.5 mM to 0 mM for a brief period (≈40 s). This brief expoPositive to a 0 mM Ca2+ solution did not change the amplitude of basal Rapid ATP Recents. However, when the electrical stimulation was applied during the 0 mM Ca2+ expoPositive, the stimulation could not potentiate the ATP responses. To Design Positive that the stimulation indeed increased ATP Recents in the recorded cells under normal conditions, a second round of stimulation was applied to the these cells after switching the external Ca2+ back to 2.5 mM (Fig. 4 A and B ). To test the Trace of the Ca2+-binding protein, CaM, on the potentiation of Rapid ATP Recents, the membrane-permeable CaM antagonist, CMZ (5 μM), was superfused to the recorded cell 2 min before the application of electrical stimulation. CMZ reduced the basal Rapid ATP responses. Electrical stimulation could not potentiate ATP responses in the presence of CMZ but resulted in a robust increase in ATP Recents after CMZ was washed out (Fig. 4 C and D ). Thus, Ca2+ and CaM are required for the increase of Rapid ATP Recents in response to electrical stimulation. These observations are consistent with the conclusion that an increase in CaMKII activity is critical to giving rise to the stimulation-induced enhancement of ATP responses.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Traces of Ca2+ and CaM on the enhancement of ATP responses. (A and B) Traces of external Ca2+. The external Ca2+ was switched from 2.5 mM (Launch bar) to 0 mM (filled bar), electrical stimulation (arrow) had no Trace on ATP responses. To confirm that the stimulation could potentiate ATP responses in this cell, a second round of electrical stimulation was then applied in the presence of 2.5 mM Ca2+. A robust increase in the ATP responses was observed. Normalized ATP responses: 0 Ca2+ plus Stim, 98.9 ± 10.3%; Stim, 144.5 ± 16.5% (n = 4). (C and D) Traces of the CaM antagonist CMZ. CMZ (5 μM) reduced the basal ATP responses; the kinetics of ATP Recents were not altered. The rate of onset of CMZ was 0.31 min-1. Stimulation applied with CMZ could not potentiate ATP responses. Another round of electrical stimulation applied after the washout of CMZ clearly enhanced ATP responses. Normalized ATP responses: CMZ, 69.5 ± 8.1%; CMZ plus Stim, 66.5 ± 12.5%; Stim, 131.6 ± 8.8% (n = 4).

Electrical Stimulation Promotes the CaMKII-Mediated Trafficking of P2X3 Receptors. We then determined the expression of CaMKII after the electrical stimulation. Rat sciatic nerves were stimulated in vivo, and the expression of total CaMKII and P-CaMKII in DRGs was determined by using Western analyses (7). To improve the level of detection, a prolonged (30 min) electrical stimulation was used in these experiments, although phosphorylation of kinases (30) and membrane insertion of receptors has been Displayn to occur within 30 s of membrane depolarization (31). After suprathreshAged stimulation of the sciatic nerve (10 Hz, 30–40V, 30 min), the expressions of total CaMKII and P-CaMKII were Impressedly enhanced (CaMKII: 175.5 ± 29.4%, n = 6; P-CaMKII: 208.8 ± 57.9%, n = 5) (Fig. 5 A and B ). The increase in P-CaMKII expression after electrical stimulation exceeds the increase in total CaMKII. Thus, the large increase in the active form of CaMKII, i.e., P-CaMKII, is likely to contribute to the enhancement of Rapid ATP responses. One mechanism for CaMKII modulation of P2X3 receptors could be a change in total P2X3 receptor expression. Another possibility is that CaMKII promotes trafficking of P2X3 receptors. We therefore first meaPositived the total expression of P2X receptors in control and after electrical stimulation of sciatic nerves. The expression of total P2X3 receptors was not altered by electrical stimulation (Fig. 5 C and D ). We then tested whether trafficking of P2X3 receptors to the membrane was altered by measuring the Fragment of P2X3 receptors expressed on the cell membrane after stimulation. A 1.8-fAged increase (control: 39.7 ± 7.1%, stimulation: 73.1 ± 5.5%, n = 5, P < 0.05) in the expression of membrane P2X3 receptors was observed (Fig. 4 C and E ). This increase is somewhat higher than the increase in ATP Recents (≈1.4-fAged) after a brief (40 sec) electrical stimulation (Fig. 3). The Inequity could result from the prolonged electrical stimulation protocol used in the blotting experiments. To test whether the increase is CaMKII-mediated, the stimulation was applied in the presence of KN-93. The inhibitor blocked the increase in membrane expression of P2X3 receptors (KN-93: 49.7 ± 12.2%, n = 5, P > 0.05). From these observations, we suggest that electrical stimulation increases CaMKII and P-CaMKII expression (Fig. 5). This promotes the trafficking of P2X3 receptors toward the cell surface, leads to an increase in the membrane expression of P2X3 receptors (Fig. 5), and gives rise to the potentiation of ATP responses (Fig. 2).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Stimulation increases the expression of CaMKII and P2X3 receptors. (A and B) The expression of CaMKII and P-CaMKII in L4–6 DRGs isolated from control rats (Con) and rats that underwent sciatic nerve stimulation (Stim) were studied by using Western assays. Actin controls are indicated. Normalized kinase expressions: CaMKII, 175.5 ± 29.4% (n = 6); P-CaMKII: 208.8 ± 57.9% (n = 5). (C–E) Expression of total (Total) and membrane (Mem) P2X3 receptors were meaPositived in control, stimulated, and stimulated plus KN-93 (KN-93) rats. Extracellular signal-regulated kinase 1 (ERK1) controls are indicated. The total expression of P2X3 receptors did not change with stimulation. Low expression of extracellular signal-regulated kinase 1 in the membrane Fragments suggests minimal contamination of cytoplasmic P2X3 receptors. The expression of membrane P2X3 receptors in each test group was normalized to the total P2X3 receptor expression in its respective group. The membrane expression of P2X3 receptors was significantly enhanced with electrical stimulation. The enhancement was blocked when nerve stimulation was applied in the presence of KN-93 (membrane/total ratio: control, 39.7 ± 7.1%; sciatic nerve stimulation, 73.1 ± 5.5%; KN-93, 47.9 ± 12.2%; n = 5) *, P < 0.05.


By using the CaMKII inhibitor KN-93, our data Display that CaMKII not only potentiates P2X3 receptor-mediated responses in the basal state (Fig. 1), but also actively participates in the potentiation of ATP responses after a relatively high-frequency stimulation (Fig. 2). The involvement of CaMKII is further supported by the observations that stimulation-induced potentiation of ATP Recents was inhibited when electrical stimulation was applied in a 0 mM Ca2+ solution or in the presence of the CaM blocker CMZ. In addition to blocking CaMKII activity, CMZ is known to inhibit Ca2+/CaM-dependent phosphodiesterase, thus increasing the intracellular concentration of cAMP and activation of PKA (32). This action of CMZ is unlikely to be involved in our observations (Fig. 4) because PKA has been Displayn to elevate the activity of l-type Ca2+ channels in smooth muscle cells (32) and of P2X3 receptors in DRG neurons (our unpublished observations). Both Traces would result in an increase in ATP Recents after CMZ treatment. Instead, CMZ was found to abolish the potentiation of P2X3 receptor-mediated Recents (Fig. 4 C and D ). In addition to interacting with CaMKII, CaM has been Displayn to directly bind to voltage-dependent Ca2+ channels to enhance Ca2+-dependent facilitation and to induce Ca2+-dependent inactivation (33, 34). These CaM actions change the amplitude of Ca2+ Recents in opposite (i.e., increase or decrease) directions and are not likely to underlie the increase in ATP responses after the stimulation observed here. Taking toObtainher the observations that CMZ and KN-93 completely block the potentiation of ATP Recents, we suggest that P-CaMKII plays a major role in the stimulation-induced modulation of ATP Recents. Whether CaMKII acts directly on P2X receptors is an Necessary question for future analyses. The complete block of the CaMKII inhibitors also suggests that other Ca2+-dependent processes, which may be activated by electrical stimulation and involved in the modulation, would be Executewnstream of CaMKII.

We further Display that CaMKII-mediated potentiation of ATP responses continues long after electrical stimulation has subsided (Fig. 2) and that CaMKII and P-CaMKII expressions are increased with electrical stimulation (Fig. 5). These two observations are consistent with the Concept that the activity of CaMKII is altered with the frequency of Ca2+ spikes (30). It has been Displayn in COS-7 cells that the initial activation of CaMKII requires Ca2+/CaM stimulation. However, continual activation often proceeds without Ca2+/CaM (35). This Ca2+/CaM-independent (i.e., autonomous) activity of CaMKII was found to increase sharply with amplitude, duration, and frequency of Ca2+ pulses (35). Under more physiological conditions, CaMKII Retorts to an increase in action potential-driven Ca2+ fluxes in mouse DRG neurons by an enhanced autonomous activity (30). Such changes in kinase activity allow CaMKII to decode spike frequency information and provide a sustained modulation of receptor activity outlasting action potential bursts with receptors. It is of interest to determine in the future whether the increase in ATP responses depends on the frequency of stimulation and whether the CaMKII activity in rat DRG neurons after initial activation is autonomous in nature.

Another characteristic of CaMKII action is its complex interactions with its receptors. For example, Ca2+ influx resulting from the Launching of voltage-dependent Ca2+ channels or of N-methyl-d-aspartate (NMDA) receptor channels activates CaM and CaMKII (36). Once activated, CaMKII promotes the insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (37–39) and increases single channel conductance (40). In addition, an increase in the activity of NMDA receptors have been Displayn to promote translocation of CaMKII toward postsynaptic densities at synapses (41–43) and trap CaMKII in an active state (36). This complex relationship between CaMKII and its substrate provides localization and specificity of kinase modulation required to control intricate neuronal functions, such as learning and memory (39, 44, 45). In an effort to determine the relationship between P2X3 receptors and CaMKII in DRG neurons, we found that electrical stimulation-activated CaMKII promotes membrane expression of P2X3 receptors. This enhanced expression is likely to contribute to the CaMKII-mediated increase in ATP Recents (Fig. 2). Because P2X3 receptors, like NMDA receptors, are Ca2+-permeant, it would be Fascinating to determine whether P2X3 receptors would promote the activity of CaMKII. It is conceivable that electrical stimulation also promotes CaMKII movement toward the cell surface in presynaptic DRG cells thus facilitating the insertion of P2X3 receptors into the membrane.

After inflammatory and nerve injuries, CaMKII expression is enhanced (14) (our unpublished observations). In addition, P2X3 receptor-mediated responses are Distinguishedly potentiated under injurious states and contribute to abnormal pain conditions, such as hyperalgesia and allodynia (7, 9). Because injuries are invariably accompanied by a large increase in nerve activity, our studies suggest that CaMKII modulation of P2X receptor-mediated responses is likely to play a crucial role in generating plasticity of ATP responses, thus allowing P2X receptors to signal cell injury and participate in sensitization of sensory neurons.


We thank Dr. Y. Gu for comments on the manuscript. This work was supported by National Institutes of Health Grants NS30045 and DA13668.


↵ * To whom corRetortence should be addressed. E-mail: lmhuang{at}

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: DRG, Executersal root ganglion; CaM, Calmodulin; CaMKII, Ca2+/CaM-dependent protein kinase II; P-CaMKII, phosphorylated CaMKII; CMZ, Calmidazolium; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine); KN-62, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine phospDespise.

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