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
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ABSTRACT |
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cardiac myocyte; calcium channel; patch clamp
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 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
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.
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MATERIALS AND METHODS |
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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'-(,
-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 3135°C by using a patch pipette (24 M) 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 5105 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.
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RESULTS |
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DISCUSSION |
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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 (
1050 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 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 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 300500 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 15721651 of the human 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
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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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