Calmodulin reverses rundown of L-type Ca2+ channels in guinea pig ventricular myocytes

Jian-Jun Xu, Li-Ying Hao, Asako Kameyama, and Masaki Kameyama

Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan

Submitted 23 February 2004 ; accepted in final form 7 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calmodulin (CaM) is implicated in regulation of Ca2+ channels as a Ca2+ sensor. The effect of CaM on rundown of L-type Ca2+ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca2+ channel activity disappeared within 1–3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 µM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca2+ concentrations ([Ca2+]); however, increase to micromolar [Ca2+] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca2+ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 µM) + ATP induced Ca2+ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5'-({beta},{gamma}-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1–10 µM) did not block the effect of CaM, indicating that the effect of CaM on the Ca2+ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca2+ channel activity, showing a cooperative effect of CaM and ATP on the Ca2+ channel. These results suggest that CaM is a crucial regulatory factor of Ca2+ channel basal activity.

cardiac myocyte; calcium channel; patch clamp


VOLTAGE-GATED L-TYPE Ca2+ channels control depolarization-induced Ca2+ entry in many cell types, triggering essential processes, such as impulse generation, muscle contraction, secretion of hormones and neurotransmitters, and gene expression (4). One of the unique characteristics of L-type Ca2+ channels is known as rundown, which is most pronounced in cells dialyzed internally in whole cell recordings or in inside-out patches (13, 24, 28). The rundown phenomenon is manifested as a disturbance in the regulation of the Ca2+ channel followed by loss of channel basal activity. Therefore, studies on rundown are important to improve our understanding of the regulation of Ca2+ channels. Although several mechanisms have been suggested for rundown, including proteolysis (3, 6, 40) and dephosphorylation (2, 7, 24, 31), the exact cause of rundown still remains unclear. We previously suggested that washout of cytoplasmic factors is the cause of rundown (20, 21). Cytoplasmic extracts added together with ATP restored channel activity to levels observed in the cell-attached mode (14, 20, 21). It has been suggested that one factor in cytoplasm is calpastatin, the endogenous inhibitor of calpain (14, 22). However, other active components in the cytoplasm involved in the recovery of Ca2+ channel rundown have not been identified.

Recently, many findings have highlighted the importance of the ubiquitous Ca2+-binding protein calmodulin (CaM) in the regulation of Ca2+ channels. It has been reported that CaM is attached to its binding domains in the carboxy-terminal tail of the {alpha}1-subunit and functions as a Ca2+ sensor for Ca2+-dependent inactivation of the channel (10, 30, 34, 35, 37, 45, 46). CaM bifurcates the local Ca2+ signal, via its amino-terminal and carboxy-terminal lobes, leading to channel inactivation and facilitation, respectively (10). Mutation of either the Ca2+-binding sites in CaM (10) or the CaM-binding domains in the carboxy terminus of the {alpha}1c-subunit (45) abolishes both Ca2+-dependent facilitation and inactivation of the channel. These findings strongly suggest the importance of CaM in the regulation of Ca2+ channels under physiological conditions. Apart from Ca2+-dependent facilitation and inactivation, however, the role of CaM in the regulation of Ca2+ channel basal activity is not clear.

In an attempt to evaluate the role of CaM in Ca2+ channel basal activity, we studied the effect of CaM on rundown of L-type Ca2+ channels in inside-out patch mode. We have found that CaM + ATP can induce activity of the Ca2+ channels after rundown and that this effect is not blocked by protein kinase inhibitors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of single myocytes. Single ventricular cells were obtained from adult guinea pig hearts by collagenase and protease dissociation as described previously (43). In brief, a female guinea pig (weight 400–600 g) was anesthetized with pentobarbital sodium (30 mg/kg ip), and the aorta was cannulated in situ under artificial respiration. The dissected heart was mounted on a Langendorff apparatus and perfused first with Tyrode solution (3 min at 37°C), then with nominally Ca2+-free Tyrode solution for 5 min, and finally with Ca2+-free Tyrode solution containing collagenase (0.08 mg/ml; Yakult) for 10–15 min. The enzyme solution was then washed out with a high-K+, low-Ca2+ solution (storage solution), and the ventricular myocytes were dispersed and filtered through a stainless steel mesh (105 µm). To improve the success rate in attaining a gigaohm seal, the myocytes were incubated in storage solution containing both protease (Nagase NK-103, 0.05 mg/ml) and DNase I (Sigma type IV, 0.02 mg/ml) for 5–10 min, after which they were washed twice by centrifugation (800 rpm for 3 min) and stored at 4°C in storage solution. The experiments were carried out after approval of the Committee of Animal Experimentation, Kagoshima University.

Solutions. Tyrode solution contained (in mM) 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1.0 MgCl2, 5.5 glucose, 1.8 CaCl2, and 10 HEPES-NaOH buffer (pH 7.4). The storage solution was composed of (in mM) 70 KOH, 50 glutamic acid, 40 KCl, 20 KH2PO4, 20 taurine, 3 MgCl2, 10 glucose, 10 HEPES, and 0.5 EGTA; pH was adjusted to 7.4 with KOH. The pipette solution contained (in mM) 50 BaCl2, 70 tetraethylammonium chloride, 0.5 EGTA, 0.003 BAY K 8644, and 10 HEPES-CsOH buffer (pH 7.4). The basic internal solution consisted of (in mM) 90 potassium aspartate, 30 KCl, 10 KH2PO4, 1 EGTA, 0.5 MgCl2, 0.5 CaCl2, and 10 HEPES-KOH buffer (pH 7.4; free Ca2+ 80 nM, pCa 7.1). CaM and ATP were dissolved in basic internal solution unless otherwise indicated. Free Ca2+ concentration ([Ca2+]) in the presence and absence of ATP was calculated with a modified computer program originally described by Fabiato and Fabiato (12). The Ca2+-free internal solution was prepared by removal of CaCl2 from the basic internal solution.

Materials. BAY K 8644 was a generous gift from Bayer (Leverkusen, Germany). CaM and MgATP were purchased from Sigma. KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide (AIP), K252a, and adenosine 5'-({beta},{gamma}-imido)triphosphate (AMP-PNP) were purchased from Peninsula Laboratories (Belmont, CA). One unit per milliliter of CaM was estimated as 1.5 nM.

Patch clamp and data analysis. Ca2+ channel activity was monitored with the patch-clamp technique. First, the cell-attached mode was formed, in which the myocytes were perfused with the basic internal solution at 31–35°C by using a patch pipette (2–4 M{Omega}) containing 50 mM Ba2+ and 3 µM BAY K 8644, a Ca2+ channel modulator. After Ca2+ channel activity was recorded, the membrane patch was excised from the cell to establish the inside-out patch configuration. For the application of CaM and ATP, the patch was moved to a small inset in the perfusion chamber, which was connected to a microinjection system. Barium currents through the Ca2+ channel were elicited by depolarizing pulses from –70 to 0 mV for 200-ms duration at a rate of 0.5 Hz. They were recorded with a patch-clamp amplifier (EPC-7; List, Darmstadt, Germany) and fed to a computer at a sampling rate of 3.3 kHz. The capacity and leakage currents in the current traces were digitally subtracted. The mean current during the period 5–105 ms after the onset of the test pulses (I) was measured and divided by the unitary current amplitude (i) to yield NPo (because I = N x Po x i), where N is the number of channels in the patch and Po is the time-averaged open-state probability of the channels.

Data are presented as means ± SE. Student's t-test was used to estimate statistical significance, and a P value <0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of CaM + ATP on Ca2+ channel activity in inside-out patch mode. We first examined the effect of CaM on Ca2+ channel activity in the inside-out patch mode in which Ca2+ channels usually showed rundown. Because ATP is required for channel activity (44), CaM was applied together with 3 mM ATP in this study unless otherwise indicated. We confirmed that channel activity decreased rapidly 1–3 min after the patch was excised to the basic internal solution. As shown in Fig. 1, A and B, application of CaM (0.03 and 0.3 µM) + ATP, at free [Ca2+] of 80 nM, within 1 min after patch excision resulted in a dose-dependent induction of channel activity [11.2 ± 5.6% (n = 7) and 94.7 ± 27.9% (n = 11) of that in the cell-attached mode, respectively]. Addition of higher concentrations of CaM (0.75 and 3 µM) + ATP induced channel activity to a level greater than that of control conditions [157 ± 99% (n = 11) and 293 ± 109% (n = 9), respectively; Fig. 1, C and D]. Figure 1D summarizes the dose-dependent effect of CaM on Ca2+ channel activity. This result suggested that CaM + ATP could prevent or reverse rundown of the Ca2+ channel.



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Fig. 1. Dose-dependent effect of calmodulin (CaM) on Ca2+ channel activity in inside-out patch mode. The channel open state probability (NPo) value for repetitive depolarization was calculated and plotted against time. After 2-min recording of NPo in cell-attached mode (ca), inside-out patch mode (io) was initiated as indicated by the arrows. Different concentrations of CaM, added together with 3 mM ATP, were then applied at the time indicated by the boxes. A–C: application of CaM at 0.03 (A), 0.3 (B), and 3 (C) µM. D: mean channel activity during 2–5 min after application of CaM (0.03, 0.3, 0.75, 3 µM) normalized to the previous activity in the cell-attached mode. Dotted line is a fitted curve with an apparent Kd of 1.06 µM, a maximum effect of 395%, and a Hill coefficient of 1.

 
It has been reported that CaM + ATP has no effect on channel activity in inside-out patches (11), seemingly in conflict with our results. It was notable that the membrane patch was excised into basic internal solution containing 80 nM free Ca2+ in our study, whereas the solution in that study contained >1 µM. To examine the possibility that [Ca2+] may alter the effect of CaM on the Ca2+ channel, the excised patch was exposed to CaM + ATP solutions containing various concentrations of free Ca2+. Figure 2A shows the results of such an experiment, in which the excised patch was first exposed to CaM (0.75 µM) + 3 mM ATP solution in the absence of free Ca2+ and channel activity was induced. Perfusion of the patch with CaM + ATP solution containing 80 nM free Ca2+ slightly increased channel activity. The open-close kinetic pattern did not seem to be different under these conditions (see, e.g., Fig. 2B). However, the induced channel activity immediately faded when the patch was perfused with CaM + ATP solution containing 1 µM free Ca2+. Figure 2C depicts the mean channel activity (NPo) reached 2–5 min after application of CaM (0.75 µM) + 3 mM ATP in the presence of different [Ca2+]. Channel activity was induced to 124 ± 26.7% (n = 6) after the excised patch was immediately exposed to CaM (0.75 µM) + ATP solution in the absence of free Ca2+. Simultaneous exposure of the excised patch to CaM + ATP solution containing 80 nM free Ca2+ induced channel activity of 157 ± 99.2% (n = 11; P > 0.05 vs. Ca2+-free condition). Elevation of free Ca2+ to 300 nM further increased channel activity to 294 ± 110% (n = 4) or rather decreased it to 5.5 ± 5.1% (n = 3), resulting in an average of 170 ± 165% (n = 7). At 1 µM Ca2+, channel activity was almost completely abolished within 10 s (0.06 ± 0.009%, n = 5; P < 0.001 vs. Ca2+-free condition), suggesting Ca2+-mediated inactivation or an acceleration of rundown. These results demonstrated a Ca2+-dependent effect of CaM on Ca2+ channels and suggested that nanomolar [Ca2+] may facilitate the effect of CaM whereas micromolar [Ca2+] exert an opposing action.



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Fig. 2. The effect of CaM on channel activity is Ca2+ dependent. A: time course of average channel activity (NPo) recorded in the cell-attached and inside-out patch modes. CaM (0.75 µM) + ATP (3 mM) containing different Ca2+ concentrations (Ca2+ free, 80 nM, and 1 µM) were applied at the times indicated by the boxes. B: single-channel traces for the corresponding experiments under the conditions indicated in A: in the cell-attached mode (a), with application of CaM + ATP in the absence of Ca2+ (b), with application of CaM + ATP at 80 nM Ca2+ concentration (c), and with application of CaM + ATP at 1 µM Ca2+ concentration (d). C: mean channel activity reached 2–5 min after exposure of the excised patch to CaM (0.75 µM)-ATP (3 mM) solution at different Ca2+ concentrations, normalized to the preceding channel activity in the cell-attached mode; n = no. of myocytes.

 
Time-dependent effect of CaM + ATP on rundown. We further investigated the effect of CaM on run-down Ca2+ channels, in which the period of rundown was changed from 2 to 10 min (Fig. 3). At the second minute after patch excision, CaM (0.75 µM) + ATP induced channel activity at a level even higher than that in the cell-attached mode (150 ± 104%, n = 9; Fig. 3A), similar to that obtained by immediate application of CaM + ATP within 1 min after inside-out patch formation (157 ± 99.2%). At the 4th, 6th, 8th, and 10th minutes after inside-out patch formation, CaM (0.75 µM) + ATP induced Ca2+ channel activity at 100 ± 27.6% (n = 5), 96.9 ± 52.3% (n = 4), 29.3 ± 17.9% (n = 4), and 16.6 ± 13.4% (n = 3) of that in the cell-attached mode, respectively (Fig. 3, B–E). Thus, after a more prolonged rundown time, the effect of CaM + ATP on channel activity became smaller (Fig. 3F), revealing that the effect of CaM on Ca2+ channel rundown was time dependent.



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Fig. 3. Time-dependent effect of CaM on Ca2+ channel activity. The NPo value for each stimulus was calculated and plotted against time. Inside-out mode was initiated at the times indicated by the arrows. CaM (0.75 µM) + ATP (3 mM) was applied at different times after inside-out formation (2, 4, 6, 8, and 10 min) as indicated by the boxes: at the 2nd (A), 4th (B), 6th (C), 8th (D), and 10th (E) minutes after patch excision. F: mean channel activity reached 2–5 min after application of CaM + ATP at the corresponding time after patch excision, normalized to the preceding channel activity in the cell-attached mode.

 
Effect of CaM requires ATP. To explore the mechanism of the effect of CaM on Ca2+ channels, the effect of CaM (0.75 µM ) or ATP (3 mM) alone on channel rundown was examined. As shown in Fig. 4, A and B, channel activity decreased to almost zero 2 min after patch excision; neither CaM nor ATP alone reactivated the channel activity, suggesting that the effect of CaM requires ATP. Furthermore, although application of CaM alone after patch excision induced channel activity of <5% of control NPo, CaM + ATP applied 8 min later induced channel activity of 136 ± 37.8% (n = 5), an extent similar to that observed for the application of CaM + ATP within 2 min (Fig. 4C). This result was not mimicked by an experiment in which ATP was applied first, followed by CaM (data not shown). We then examined the dose-dependent effect of ATP in the presence of CaM. As shown in Fig. 5, A and B, 500 µM ATP + CaM (0.3 µM ) induced minimal channel activity. After replacement with 1 mM ATP, the channel activity was reactivated. Figure 5C shows a summary of the average channel activity induced by CaM (0.75 µM) + ATP at different concentrations (0, 0.5, 1, and 3 mM) applied within 1 min after patch excision. In the absence of ATP or in the presence of 0.5 mM ATP, CaM had almost no effect on channel activity after rundown [2.6 ± 1.9% (n = 7) and 3.7 ± 2.1% (n = 3), respectively], whereas in the presence of 1 and 3 mM ATP, channel activity was induced to 57 ± 39.1% (n = 4) and 157 ± 99.2% (n = 11), respectively. This result indicated that millimolar concentrations of ATP were required for CaM to be effective.



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Fig. 4. Effect of CaM on Ca2+ channel activity requires ATP. The NPo value for repetitive depolarization was calculated and plotted against time in the cell-attached and inside-out modes. A and B: 2 min after inside-out configuration formation, application of CaM (0.75 µM) or ATP (3 mM) alone, respectively, had no effect on channel activity. C: CaM (0.75 µM) alone was first applied when the patch was excised, followed 8 min later by CaM (0.75 µM) + ATP (3 mM) as indicated by the boxes. The channel activity induced was comparable to that produced by the application of CaM + ATP within 2 min after patch excision.

 


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Fig. 5. The effect of CaM requires millimolar ATP. A: time course of mean average channel activity (NPo) in the cell-attached mode followed by inside-out patch configuration before and after application of CaM (3 µM) + ATP (0.5 or 1 mM). B: consecutive current traces for the corresponding experiments under the conditions indicated in A: cell-attached mode (a), inside-out patch configuration (b), application of CaM (3 µM) + ATP (0.5 mM) (c), and application of CaM (3 µM) + ATP (1 mM) (d). C: mean channel activity in the presence of CaM (0.75 µM) + ATP, normalized to the preceding channel activity in the cell-attached mode; n = no. of myocytes. CaM had no effect on the channel activity in the absence of ATP or in the presence of 0.5 mM ATP, whereas in the presence of mM ATP (1 and 3 mM), CaM significantly induced channel activity, showing that millimolar ATP is required for the effect of CaM.

 
Effect of CaM is not due to phosphorylation. Because MgATP is a substrate for protein phosphorylation and it has been reported that protein kinases recover channel activity from rundown (2, 7, 24, 31), CaM might have exerted its effect by enhancing the phosphorylation of Ca2+ channels. In accordance with this, Dzhura et al. (11) and Wu et al. (42) reported that CaMKII facilitated Ca2+ current (ICa) and sustained channel activity at a high level in the inside-out patch mode. We therefore investigated whether phosphorylation mediated the CaM effect. As shown in Fig. 6, we examined the effect of CaM on channel activity in the presence of different types of CaMKII inhibitors, such as KN-62 (which inhibits CaMKII by preventing binding of CaM to the kinase), a specific CaMKII-inhibitory peptide CaMKII 281-309 (which inhibits CaMKII by blocking binding of CaM and ATP to the kinase), and AIP (which acts as a pseudosubstrate). None of these inhibitors (each 1–10 µM) blocked the effect of CaM (Fig. 6), suggesting that the effect of CaM was not mediated by phosphorylation by CaMKII. We note that KN-62 and its derivative KN-93 have been reported to block the whole cell ICa directly, as a side effect in addition to inhibiting CaMKII (1, 36). In the present study, however, KN-62 had no inhibitory effect on Ca2+ channel activity induced by CaM, suggesting that KN-62 (10 µM) had little or no directly blocking action on the Ca2+ channel in the inside-out patch mode.



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Fig. 6. The effect of CaM on Ca2+ channel activity is not mediated by CaM-dependent protein kinase (CaMK)II. Time course of average channel activity (NPo) in the cell-attached and inside-out modes with application of CaM (0.75 µM) + ATP (3 mM) is shown. The effect of CaM on channel activity was not blocked by the various types of inhibitors of CaMKII: KN-62 (10 µM; A), CaMKII inhibitor peptide CaMKII 281-309 (10 µM; B), autocamtide-related CaMKII peptide (AIP, 10 µM; C). D: summary of the mean channel activity in the presence of CaMKII inhibitors. There was no significant difference between the control and any inhibitor.

 
We then examined whether any other protein kinases were involved in the effect of CaM. As shown in Fig. 7A, the broad protein kinase inhibitor K252a (1–10 µM) did not block the effect of CaM. This result suggested that the action of CaM on Ca2+ channels was phosphorylation independent. In addition, CaM added together with AMP-PNP, a nonhydrolyzable ATP analog, also induced channel activity, although the effect was relatively small (21.6 ± 7.4%, n = 9) and tended to decline with time (Fig. 7, B and C). Similar results were obtained with CaM + {beta},{gamma}-methyleneadenosine 5'-triphosphate (AMP-PCP; 17.3 ± 5.6%, n = 6) and CaM + GTP (4.5 ± 2.8%, n = 6) (data not shown).



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Fig. 7. The effect of CaM is not mediated by phosphorylation. A: effect of CaM (0.75 µM) + ATP (3 mM) was not blocked by the nonspecific protein kinase inhibitor K252a (10 µM). B: CaM + adenosine 5'-({beta},{gamma}-imido)triphosphate (AMP-PNP; 3 mM) induced channel activity with a lower potency and a shorter duration. C: summary of mean channel activity for control (CaM + ATP), K252a (CaM + ATP + K252a), and AMP-PNP (CaM + AMP-PNP). There is no significant difference between the control and K252a groups, whereas there is a significant difference between the control and AMP-PNP groups (**P < 0.01).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we have characterized the effect of CaM on the activity of L-type Ca2+ channels in inside-out patch mode. The most important finding is that exogenously applied CaM restores Ca2+ channel activity in a rundown state under cell-free conditions.

Dynamic interaction of CaM with Ca2+ channel. CaM is a ubiquitous and major Ca2+-binding protein, which binds to and regulates various ion channels, including L- and P/Q-type Ca2+ channels, voltage-gated Na+ channels, SK K+ channels, and ryanodine-sensitive Ca2+ release channels in sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) (for reviews see Refs. 19 and 41). CaM exists in cells in two forms, apoCaM and Ca2+/CaM (including Ca2+-unsaturated and Ca2+-saturated CaM) (for reviews see Refs. 19 and 33). It has been suggested that apoCaM is preassociated with L-type Ca2+ channels and functions as a Ca2+ sensor for Ca2+-dependent inactivation and facilitation of the channel. CaM-mediated Ca2+-dependent inactivation and facilitation have been well characterized, but the effect of CaM on Ca2+ channel basal activity remains unclear.

In this study, we found that CaM reactivated rundown channels in inside-out patch mode, in a dose-dependent manner with a threshold concentration of ~30 nM. The presence of ATP is required for CaM to be effective. These results suggest that binding of CaM to the channel in the presence of ATP is required for the basal activity of the channel. If we assume that the dose-response relation (Fig. 1D) simply reflects the binding of CaM to the channel, the Kd for CaM binding would be ~1 µM, which is much larger than that reported for Ca2+/CaM binding to fragments of carboxy-terminal regions of the channel (~10–50 nM; Refs. 30 and 46). Thus the results imply that the association of apoCaM with the channel is not so tight but may be in a dynamic equilibrium with free apoCaM in the cytoplasm near the internal side of the channel. CaM at ~0.3 µM + ATP (3 mM) produced channel activity comparable to that seen in the preceding cell-attached mode (Fig. 1), suggesting that the concentration of free CaM near the Ca2+ channel in the myocytes is in this range. We speculate that only one-third of the channels are bound to CaM. CaM at >0.3 µM (+ 3 mM ATP) produces channel activity higher than that seen in the cell-attached mode. This suggests a new mechanism of channel modulation: changes in the concentration of free CaM by release from or absorption to CaM-binding proteins may modulate activity of the Ca2+ channel.

The effect of CaM on channel activity is Ca2+ dependent: the channel is activated by CaM at [Ca2+] greater than ~100 nM but is inhibited by Ca2+ at micromolar levels. This result implies that interaction of apoCaM or Ca2+-unsaturated CaM with the channel is necessary for activation of the channel and that the effect of Ca2+-saturated CaM may be Ca2+-dependent inactivation of the channel. Similar findings have been reported for the ryanodine receptor (Ca2+ release channel in SR) in skeletal muscle: CaM acts as an activator at nanomolar [Ca2+] but as an inhibitor at micromolar [Ca2+] (38, 39).

Mechanism of CaM effect on Ca2+ channel activity. Apart from the direct interaction of CaM with the Ca2+ channel, it has also been suggested that CaM influences channel activity indirectly through activation of CaMKII, phosphodiesterase (PDE) type 1, or calcineurin (CaM-dependent protein phosphatase) (for reviews see Ref. 33). In particular, it has been reported that CaMKII-mediated phosphorylation of the channel enhances channel activity, contributing to Ca2+-mediated facilitation of the channel (11, 27, 42). Thus it was necessary to assess whether the effect of CaM on the Ca2+ channel is mediated by CaMKII.

Because CaMKII 281-309 blocks Ca2+-dependent binding of CaM to CaMKII (29), our result excludes the possibility that the CaM effect is mediated by Ca2+/CaM-dependent activation of CaMKII. Contribution of basal activity of CaMKII is also unlikely because other types of CaMKII blockers, i.e., KN-62 (inhibitor of CaM binding to CaMKII) and AIP (pseudosubstrate of CaMKII), have no effect as well. Because KN-62 has been suggested to directly block the whole cell Ca2+ current in addition to its inhibition of CaMKII (1, 36), the effect of KN-62 should be evaluated carefully. However, as noted in RESULTS, the negative effect of KN-62 (10 µM) on Ca2+ channel activity induced by CaM in excised patches indicates that KN-62 at this concentration does not directly block the Ca2+ channel from the intracellular side. In conclusion, the result that various inhibitors of CaMKII (KN-62, CaMKII 281-309, and AIP) do not inhibit the CaM effect indicates that the CaM effect is not mediated by CaMKII and thereby supports the view that a mechanism underlying the CaM effect is different from that for CaMKII-mediated facilitation.

Furthermore, a nonspecific inhibitor of protein kinases (K252a) did not alter the effect of CaM, suggesting that mediation by other protein kinases is also unlikely. Thus our results support the view that CaM interacts directly with the Ca2+ channel and reverses the rundown. This view is in line with the recent proposal that CaM associates with the Ca2+ channel and regulates its activity (25, 26, 30, 35). Disruption of the binding of CaM to the channel by mutation of the CaM-binding domain in the carboxy-terminal tail of the {alpha}1C-subunit results in a loss of regulatory effect of CaM (10, 45). Direct association of CaM with channel proteins has also been reported in the P/Q-type Ca2+ channel, ryanodine receptor, Ca2+-activated K+ channels (SK and IK type), ether à go-go (EAG)-type K+ channel, and voltage-gated Na+ channel (for review see Refs. 19 and 41).

Possible involvement of protein kinases. The effect of CaM requires millimolar concentrations of ATP. As discussed above, the involvement of protein phosphorylation in the effect of CaM + ATP is unlikely. A further supporting result for this view is that ATP can be replaced partially by AMP-PNP, a nonhydrolyzable ATP analog. Because ATP alone had no effect on channel activity, a phosphorylation-independent action of ATP may be involved in the action of CaM (32, 44).

In cardiac L-type Ca2+ channels, regulation of the channel by protein phosphorylation mediated by cAMP-dependent protein kinase A (PKA) is well documented (16). PKA is anchored to its anchoring protein (AKAP) near the channels (8), whereas protein phosphatase 2A (PP2A) binds directly to the carboxy-terminal tail of the {alpha}1c-subunit (9), providing spatial and temporal coordination of channel regulation. Thus Ca2+ channel activity is thought to be balanced by phosphorylation by PKA and dephosphorylation by PP2A. In GH3 cells, rundown of Ca2+ channels is prevented or reversed by dialyzing the cells with cAMP or PKA (2). A similar conclusion has also been reported for the cardiac L-type Ca2+ channel. However, Yazawa et al. (44) reported that recovery of cardiac Ca2+ channel activity from rundown is essentially independent of PKA. In the present study, we found that PKA + ATP or okadaic acid + ATP are capable of maintaining channel activity to only a small extent in the inside-out patch configuration. Compared with the enormous effect of CaM + ATP on channel activity, it is likely that phosphorylation alone is not sufficient for recovery of the channel from rundown.

One finding relevant to this point is that the effect of CaM + ATP on the channel is time dependent: it is attenuated by increasing the period of rundown and finally abolished when the duration of rundown is >10 min (Fig. 3F). This result implies that the channel changes with time from a CaM-responsive state (early phase) to a nonresponsive state (late phase) in the inside-out configuration. It is interesting to note that the putative CaM-responsive state seems to be stabilized by CaM itself (see Fig. 4C), implying a CaM-mediated conformation change of the channel protein. Thus the hypothesis that the dynamic interaction of CaM with the channel proteins is modified by other regulatory mechanisms, such as phosphorylation of the channel, may be worth future investigation.

CaM is involved in channel rundown in inside-out patch mode. So far, the exact cause of rundown remains unclear. Several mechanisms have been suggested to explain the cause of rundown, including proteolysis (3, 6, 40) and dephosphorylation (2, 17, 24, 31). In addition, there is evidence that several factors are involved in rundown, including Ca2+, ATP, calpastatin, and an unidentified factor in cytoplasm (4, 14, 17, 22). Rundown seen in the inside-out patch configuration is reversible and is not prevented by protease inhibitors, suggesting that rundown is not due to proteolysis. Also, reversal of rundown by PKA or CaMKII is controversial.

In the present study, we found that CaM completely prevents or reverses the rundown of L-type Ca2+ channels in inside-out patch mode, suggesting that CaM is a crucial regulatory factor for the maintenance of Ca2+ channel basal activity. Interestingly, the effect of CaM requires millimolar ATP and a low [Ca2+] (less than ~300–500 nM). Thus, in intact cells, a reduction in ATP or an increase in free Ca2+ in the cytoplasm would result in attenuation of the CaM effect on the channel activity. This idea is consistent with previous findings in whole cell recordings that the ICa is maintained by dialyzing the cells against ATP or Ca2+ chelators (EGTA or BAPTA) (2, 17). It can be speculated that ATP and low levels of Ca2+ might be important for maintaining the conformation of CaM and the channel, respectively, to be capable of interaction with each other to produce the basal activity of the channel.

We have previously proposed (20, 22, 40) that calpastatin, an endogenous inhibitor of Ca2+-activated protease calpain, is involved in the maintenance of basal activity of the L-type Ca2+ channel. Although application of calpastatin or its effective fragments + ATP restores channel activity in the inside-out configuration, its effect is much smaller than that of CaM + ATP (14, 15). It should be stated, therefore, that calpastatin is probably not the major component that interacts with the Ca2+ channel and primes it for voltage-dependent activation. Nevertheless, the relationship between the CaM effect and the effects of calpastatin and proteins in the cytoplasm in terms of regulation of Ca2+ channel activity should be clarified in further investigations.

Proposed mechanism of rundown. On the basis of both previous and present findings, we suggest that the mechanism of rundown of cardiac L-type Ca2+ channels in inside-out patch mode is as follows. The channels in intact cells are under the influence of the dynamic interaction of cytoplasmic CaM with the binding site in the channels. When the cytoplasm is washed out by excision of the membrane patch, the channel undergoes rundown due to release of CaM and/or loss of ATP. In this condition, the rundown can be prevented by supplementing with CaM and ATP. However, the conformation of the channel gradually changes in such a way that repriming by CaM and ATP is attenuated, possibly because of dephosphorylation of a certain site of the channel. Thus there may be two states of rundown: one is reversible by CaM + ATP (early phase) and the other is not (late phase).

Recently, Kepplinger et al. (23) reported that the carboxy-terminal sequence 1572–1651 of the human {alpha}1c-subunit, which contains two CaM binding domains (CBD and IQ), is also a target site for calpastatin and thus the major region for the run-down property of the channel. Another study has revealed a third CaM binding site located in the amino-terminal tail of the {alpha}1c-subunit, which also contributes to the Ca2+/CaM-dependent inactivation of the channel (18). Thus it is important to test which one of the CaM-binding sites is important in maintaining the basal activity of the channel.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by grants from the Japan Society for the Promotion of Science, the Kodama Memorial Foundation (to M. Kameyama), the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Inamori Foundation (to L-Y. Hao).


    ACKNOWLEDGMENTS
 
We thank T. Imaichi, E. Minobe, and Y. Komatsu for technical assistance and E. Iwasaki for secretarial work on the manuscript. J.-J. Xu thanks Professor W.-J. Fu and Professor K. Xu for encouragement.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Kameyama, Dept. of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima Univ., Sakuragaoka, Kagoshima 890-8544, Japan (E-mail: kame{at}m.kufm.kagoshima-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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