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Department of Neurobiology, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel
Submitted 21 April 2008; accepted in final form 2 August 2008
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ABSTRACT |
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INTRODUCTION |
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) are highly conserved and function to posttranslationally modify substrates by partial proteolysis rather than by complete proteolytic digestion (Carragher and Frame 2002; Evans and Turner 2007). Calpains are known to proteolyze >100 substrates that lack strong sequence homology in various tissues. Consequently, the calpain superfamily has been implicated in playing a central role in diverse physiological and pathological processes (Bevers and Neumar 2008; Evans and Turner 2007; Goll et al. 2003; Wu and Lynch 2006). For example, calpains play physiological roles in cell motility (Perrin and Huttenlocher 2002), apoptosis (Squier et al. 1994), cell differentiation (Simonson et al. 1985), and synaptic transmission (for a review, see Wu and Lynch 2006). On the other hand, calpains are also thought to play roles in the pathogenesis of Huntington's, Parkinson's, and Alzheimer's diseases, cataract formation, diabetes, ischemic and traumatic brain injury, the pathogenesis of stroke, myocardial infarction, and muscular dystrophy (Bevers and Neumar 2008; Higuchi et al. 2005; Lescop et al. 2005; Ray et al. 2003; Saatman et al. 1996). Understanding the physiological roles of calpains, on the one hand, and the potential use of calpain inhibitors in preventing pathological processes, on the other, led to the development of a large number of calpain inhibitors (Bevers and Neumar 2008; Ray 2006; Ray et al. 2003; Saez et al. 2006).
Interpretation of the experimental results obtained by using these inhibitors and the translation of these results to clinical applications are complicated by the fact that calpains cleave multiple substrates and that many calpain inhibitors also inhibit other cysteine proteases, serine proteases, and the proteasome (Barrett et al. 1982; Wang and Yuen 1994).
In a series of earlier studies, we found that a transient elevation of intracellular calcium ion concentration ([Ca2+]i) in cultured Aplysia neurons, after axotomy, activates cytoplasmic proteases. Since the proteolytic activity was induced by a transient elevation of [Ca2+]i, blocked by the membrane-permeable calpain inhibitor, calpeptin (Tsujinaka et al. 1988), and cleaved the classical calpain substrate, spectrin, we functionally classified this proteolytic activity as generated by calpain (Gitler and Spira 1998, 2002; Khoutorsky and Spira 2005; Sahly et al. 2006). While studying the role of calpain in synaptic facilitation of cultured Aplysia sensorimotor synapses, we noticed that inhibition of calpain initially increases evoked neurotransmitter release (Khoutorsky and Spira 2005). The initial increase in neurotransmitter release was associated with broadening of the presynaptic action potentials. Therefore in the present study we undertook to examine the effects of calpain inhibitors on the excitable membrane properties.
We found that bath application of the calpain inhibitors calpeptin, calpain inhibitor XII, and MG132 leads to spike broadening and increased calcium influx due to a significant reduction of the inactivating and noninactivating voltage-gated potassium conductances. Consistent with recent reports (Suzuki et al. 2002; Wu et al. 2005; Yuen et al. 2007), our observations suggest that constitutive calpain activity regulates the properties of integral membrane proteins. The above-cited observations should be taken into account when considering the physiological roles of calpains as "housekeeping" proteases and when evaluating the use of calpain inhibitors as neuroprotective drugs.
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METHODS |
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Left upper quadrant (LUQ) neurons from the abdominal ganglia of Aplysia californica were isolated and maintained in culture, as described by Schacher and Proshansky (1983). Briefly, animals were anesthetized by injection of isotonic MgCl2 solution. The ganglia were isolated and incubated for 1.5–3 h in 1% protease (type IX, Sigma) at 34°C. Next, the ganglia were desheathed and the cell bodies of their neurons with their axons were pulled out with sharp micropipettes and placed on poly-L-lysine-coated (Sigma) glass-bottom culture dishes. The culture medium consisted of 10% filtered hemolymph from Aplysia faciata, collected along the Mediterranean coast, and L-15 (Gibco-BRL) supplemented for marine species. By 24 h after plating, the dishes were transferred to an 18°C incubator. The experiments were performed 1–2 days after plating, at room temperature (between 20 and 24°C), after replacing the culture medium with artificial sea water (ASW).
Electrophysiology
For current-clamp experiments, LUQ neurons were impaled by a sharp 5- to 10-M
glass microelectrode filled with 2 M KCl. The microelectrode served for both current injection and voltage recording (Axoclamp-2A; Axon Instruments). The membrane potential was kept at –50 mV by passing DC. Single spikes were generated by 5- to 10-ms-long depolarizing current pulses. At the end of the experiments, the stimulation intensity was gradually reduced until no spike was elicited and the subthreshold stimulus artifact was subtracted from the signal.
Voltage-gated ionic currents were analyzed using conventional two-electrode voltage clamp. For these experiments, the main axon of the neuron was trimmed off about 20 min prior to the experiment with the sharp tip of a micropipette under visual control, as previously described (Benbassat and Spira 1993; Hasson et al. 1993, 1995). In most cases this procedure enabled better space clamping. The cell body of a cultured neuron was impaled by two microelectrodes: one for current injection and the other for voltage recordings. Current records were corrected by subtracting the linear components of leak and capacitative currents.
Solutions
Control experiments were carried out in ASW composed of (in mM) 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, and 10 HEPES [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; Sigma], adjusted to pH 7.6.
To isolate the sodium current, we used a solution composed of (in mM) 410 NaCl, 10 CsCl, 65 MgCl2, 10 HEPES, 50 TEA (tetraethylammonium chloride), 0.1 3,4-DAP (3,4-diaminopyridine), and 1 Co. The pH was adjusted to 7.6.
To isolate potassium currents, we blocked the sodium and calcium currents by a solution composed of 460 mM NaCl, 10 mM KCl, 65 mM MgCl2, 10 mM HEPES, 1 mM Co, and 200 µM tetrodotoxin (TTX, Alomone labs, Jerusalem, Israel). The pH was adjusted to 7.6.
To inhibit potassium current, we used the potassium channel-blocking solution (PCBS) composed of (in mM) 410 NaCl, 10 CsCl, 11 CaCl2, 55 MgCl2, 50 TEA, 0.1 3,4-DAP, and 10 HEPES, adjusted to pH 7.6.
Calcium current was isolated using a solution composed of (in mM) 460 TEA, 1 MgCl2, 10 CsCl, 65 CaCl2, and 10 HEPES. The pH was adjusted to 7.6.
Drugs
Calpeptin, MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal), and calpain inhibitor XII (Calbiochem) were prepared as 50 mM stock solutions in dimethyl sulfoxide (DMSO; Sigma) and diluted to the final concentration just before the experiments. clasto-Lactacystin β-lactone (Calbiochem) was prepared as 10 mM stock solutions in DMSO. In control experiments, bath application of a vehicle solution composed of 0.2% DMSO in ASW had no effect on the action potential shape.
Calcium imaging
The system used for confocal imaging consisted of an Olympus microscope IX70 and a Bio-Rad (Hercules, CA) Radiance 2000/AGR-3 confocal imaging system. The objective used was an Olympus planApo x60 1.4 numerical aperture oil objective. The images were collected and processed by using LaserSharp and LaserPix Bio-Rad software, respectively. For calcium imaging, 10 mM fluo-4 (pentapotassium salt; Invitrogen) in 2 M KCl was loaded into the neurons by pressure injection. Imaging was performed after the dye had equilibrated throughout the main axon (
30 min). The dye was excited by a 488-nm laser line and the emitted lights were collected at 500–560 nm.
Imaging of proteolytic activity
Neurons were continuously incubated in ASW containing 10 µM bis(CBZ-alanyl-alanine amine)-rhodamine 110 (bCAA-R110; Molecular Probes) (Leytus et al. 1983) and were confocally imaged to determine the relative levels of proteolytic activity as previously described (Gitler and Spira 1998). The effect of MG132 and calpain inhibitor XII on the axotomy-induced increase in the bCAA-R110 fluorescent signal was tested (Gitler and Spira 1998). Whereas MG132 (100 µM, n = 7) and calpain inhibitor XII (100 µM, n = 8) prevented the elevation of the bCAA-R110 signal, clasto-lactacystin β-lactone (10 µM, n = 5) had no significant effect. In control experiments, bath application of a vehicle solution composed of 0.2% DMSO in ASW had no effect on calcium-activated cleavage of the proteolytic substrate.
Statistics
All the data are presented as means ± SE. For all statistical analyses of significance, the paired Student's t-test was used.
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RESULTS |
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Bath application of the calpain inhibitor calpeptin (100 µM) led, within 5 min, to an increase in spike duration and a slight attenuation of its amplitude. In most of the experiments (71%, n = 30/42 experiments), applying calpeptin resulted in the appearance of a second low-amplitude delayed spike (Fig. 1, A–C). In the remaining experiments (29%, n = 12/42), calpeptin resulted in decreased afterhyperpolarization (AHP) as well as spike broadening without the appearance of the delayed spike (Fig. 1E). When a train of action potentials was generated by a 1-s-long intracellular depolarizing pulse, the time interval between the first spike and the delayed one was reduced, until the second action potential fused with the falling phase of the first action potential (Fig. 1C). Following a wash, the shape of the action potential recovered within 5–10 min (Fig. 1, D and E, wash).
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We began to investigate these cited possibilities by testing the hypothesis that the second action potential, generated by a single stimulation after applying calpeptin, is a "back reflection" of a spike initiated at an electrically remote location. To that end, we decreased the electrical length of the neuron by cutting away a large part of the axon, leaving the cell body and a short segment of the main axon intact. After a rest period of about 30 min, the neuron recovered from the transection (Benbassat and Spira 1993; Spira et al. 1993) and became isopotential (Hasson et al. 1995). Since under these conditions a second spike was still generated in response to a single stimulus in the presence of calpeptin, we concluded that it represents alterations in voltage-gated currents (n = 7).
To differentiate between the possibilities that the second low-amplitude spike (that in some experiments fuses with the first spike and appears as a prolonged "shoulder" on the falling phase of the first spike) is generated by voltage-gated calcium channels or by latent sodium conductance, we applied 200 µM TTX to neurons that fired broadened action potentials in the presence of calpeptin (Fig. 2, n = 5). This led to blockade of the first peak (which is generated by inward sodium current) but not the prolonged shoulder, suggesting that the "shoulder" is generated either by voltage-gated calcium channels or by TTX-insensitive sodium channels (Yoshida 1994). Since the shoulder was blocked by a solution in which CaCl2 was replaced by MgCl2, and 1 mM cobalt (Fig. 2D), we concluded that the "shoulder" as well as the second action potential are generated by calcium currents. Consistent with this conclusion, we found that spikes generated in the presence of calpeptin result in a larger increase in the free intracellular calcium levels ([Ca2+]i) than in controls. For these experiments, neurons were loaded with fluo-4 and the changes in the [Ca2+]i were measured by confocal microscopy in the line-scan mode, as previously described by our laboratory (Malkinson and Spira 2006). We found that the [Ca2+]i is elevated to a higher value in the presence of calpeptin than in the control (Fig. 3, n = 4). The increase in the [Ca2+]i was observed only after broadening of the spike. This temporal correlation suggests that the elevation in [Ca2+]i represents increased calcium influx due to spike broadening. However, we cannot rule out the possibility that a fraction of the elevated [Ca2+]i represents calcium release from intracellular stores.
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; Lee and Goldberg 1998), calpain inhibitor XII (Li et al. 1996), and the specific proteasome inhibitor clasto-lactacystin β-lactone (Dick et al. 1996; Fenteany et al. 1995). We found that MG132 (100 µM, n = 6; 200 µM, n = 5) and calpain inhibitor XII (100 µM, n = 5; 200 µM, n = 4) decreased the hyperpolarizing afterpotential, reduced the peak amplitude of the action potentials, generated a delayed spike in a fraction of the experiments (200 µM MG132, three of five and calpain inhibitor XII two of four, not shown), and broadened the action potentials (Fig. 4, B and C, left) in a manner similar to that of calpeptin. As in the case of calpeptin, the effects of MG132 and calpain inhibitor XII reversed after wash. In contrast, a 30-min incubation of the neurons in the proteasome inhibitor clasto-lactacystin β-lactone (10 µM, n = 6) had no significant effect on the action potential waveform (not shown).
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To gain better insights into the mechanisms by which the calpain inhibitors alter the action potential shape, we analyzed the underlying voltage-gated ionic currents using the standard two-electrode voltage-clamp method (Hasson et al. 1995; McIntosh et al. 1995). In all experiments, the neurons were depolarized from a holding potential of –50 mV to various potentials.
Recording the overall macroscopic currents revealed that 100 µM calpeptin decreases both the inward and outward currents within 5 min of application (Fig. 4A, right, n = 7). Removing the calpeptin by washing resulted in partial recovery of the currents.
The overall effects of MG132 (n = 6) and calpain inhibitor XII (n = 5) on the inward and outward currents were similar to those of calpeptin (compare Fig. 4, A, right and B and C, right). clasto-Lactacystin β-lactone (10 µM) had no effect on inward and outward voltage-gated currents (not shown, n = 4).
To examine the effect of calpeptin on IK+, INa+, and ICa2+, we systematically blocked two ion currents while measuring the third one.
To study the effects of calpeptin on potassium currents (Fig. 5), we blocked INa+ and ICa2+ (see METHODS). Under these conditions, application of calpeptin resulted in a significant decrease in IK+ (Fig. 5A; compare control and calpeptin, n = 5). Whereas the mean early inactivating IK+, which lasts about 20 ms, from the onset of the voltage-clamp step, decreased by 79% when the membrane potential was stepped to +25 mV (P < 0.05, open arrow in Fig. 5, A and B), the delayed noninactivating IK+ decreased by 87% (P < 0.01, dark arrow in Fig. 5, A and C).
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We next examined the effects of calpeptin on INa+ (Fig. 7). To that end, ICa2+and IK+ were blocked by replacing all potassium ions by cesium and all calcium ions by magnesium (see METHODS). In addition, the solution contained 50 mM TEA, 0.1 mM 3,4-DAP, and 1 mM cobalt. Under these conditions, in addition to the classical early inward INa+, we also measured a residual noninactivating outward current (Fig. 7Ab).
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To examine whether calpeptin alters the inward calcium current (Fig. 8), we blocked INa+ and IK+ (see METHODS). Under these conditions, in the current-clamp mode, a short stimulus generates a long-lasting calcium action potential. Application of calpeptin shortened the calcium spike (compare Fig. 8A, control and calpeptin, left, n = 4). Voltage-clamp experiments revealed that calpeptin slightly reduces the peak ICa2+ (11%, P < 0.05, n = 4; Fig. 8, A, right and B).
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DISCUSSION |
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Voltage-clamp analysis revealed that applying calpeptin results in 1) a significant reduction in the early inactivating and the noninactivating potassium currents, 2) blockade of a residual noninactivating outward current that is not blocked by the potassium and calcium currents blocking solution used in this study, 3) a moderate (36%) reduction in the sodium current, and 4) a small 11% decrease in the calcium current. The reduction in these currents is not associated with significant alterations in the sodium current–voltage relationships and its steady-state inactivation properties. The kinetics of the blockade of potassium currents by the calpain inhibitors is sufficient to account for the appearance of the delayed spike that fuses with the first spike (Fig. 1). Under control conditions, the voltage-dependent potassium conductances are sufficient to prevent the development of a second spike, or spike broadening. Following the application of the calpain inhibitors, the noninactivating potassium conductance is reduced, allowing for the development of a second calcium-dependent spike. Initially, the early inactivating potassium conductance (that lasts for about 20 ms) repolarizes the membrane potential, terminates the first spike, and prevents the calcium conductance from contributing to the falling phase of the spike. Nevertheless, the calcium conductance is sufficient to generate a second spike when the early inactivating potassium conductance is terminated. With time, when the early inactivating potassium conductance is also reduced by the calpain inhibitors, the second spike fuses with the falling phase of the first spike, generating the "shoulder." The fact that in a fraction of the experiments a second spike was not observed is attributed to variability in the rates and levels to which the early inactivating potassium conductances were reduced.
Possible mechanisms by which calpain inhibitors alter the ionic conductances
Theoretically, the reduction in the voltage-gated potassium and sodium conductances by the calpain inhibitors could be attributed to two principal mechanisms: 1) alterations in the voltage-gated potassium and sodium currents by inhibition of calpain-mediated proteolysis and 2) via binding and blockage of the ion channels by the inhibitors.
When considering the first hypothesis, it should be noted that inhibition of IK+ and INa+ by the calpain inhibitors takes place following incubation of resting, nonstimulated neurons, in which the [Ca2+]i, at the bulk of the axoplasm, is about 100 nM (Ziv and Spira 1993). Despite the very low [Ca2+]i, constitutive cleavage of a fluorogenic proteolytic substrate, bCAA-R110, was imaged (Gitler and Spira 1998). The constitutive calpain activity may represent compartmentalized submembrane domains in which the [Ca2+]i is high in the vicinity of calcium channels (Berridge 2006). Thus theoretically, the alterations in the excitable membrane properties could be attributed to inhibition of constitutive calpain activity.
The onsets of IK+ and INa+ inhibition by calpeptin were detected by voltage-clamp measurements within <1 min of calpeptin application (Fig. 9). The rapid onset of the inhibition may indicate that the calpain inhibitors directly block the channels. Nevertheless, on-line imaging of the proteolysis of an exogenous proteolytic calpain substrate, bCAA-R110, in axotomyzed cultured Aplysia neurons revealed that the onset of calpain inhibition by bath application of calpeptin is detected within <1 min of application (Gitler and Spira 2002). Thus based on the onset time for inhibition, we cannot favor one hypothesis over the other. The rapid recovery of the channel functions after calpeptin washout also corresponds to the rapid recovery of proteolytic activity after calpeptin removal, as corroborated by images of bCAA-R110 in transected axons (Gitler and Spira 2002). Thus this parameter also cannot be used to elucidate the above-cited possibilities.
Another parameter that we considered is the correlation between the effects of the inhibitors on the ionic conductances and their effects on the proteolysis of the fluorogenic proteolytic substrate bCAA-R110. Whereas calpeptin, calpain inhibitor XII, and MG132 inhibit IK+ and INa+ and inhibit the proteolysis of bCAA-R110 (see METHODS and Gitler and Spira 1998), the proteasome inhibitor clasto-lactacystin β-lactone did not induce spike broadening and did not block the cleavage of bCAA-R110. These observations support the hypothesis that inhibition of calpain modulates the channels’ conductances.
Generalization of the present findings
Inhibition of proteolytic activity, as indicated by the use of the fluorogenic substrate bCAA-R110 in cultured Aplysia neurons, is almost total at a concentration of 100 µM calpeptin (Gitler and Spira 1998, 2002). This concentration also blocks the supply of vesicles to the readily releasable store in sensorimotor synapses of cultured Aplysia neurons (Khoutorsky and Spira 2005). Here we found that 100 µM calpeptin blocks about 80% of the potassium conductance (Fig. 5) and 36% of the sodium conductance (Fig. 7).
A comparable analysis of the effects of calpain inhibitors on excitable membrane properties in vertebrate neurons is not available. It is therefore not known whether similar alterations in the properties of ion channels are induced by calpain inhibitors and, if so, at what concentrations. Since the potential use of calpain inhibitors as protecting drugs is extensively considered, it would be beneficial to examine its effects on vertebrate neurons and cardiac muscles.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Spira, Department of Neurobiology, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel (E-mail: spira{at}cc.huji.ac.il)
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  A. Khoutorsky and M. E. Spira Activity-dependent calpain activation plays a critical role in synaptic facilitation and post-tetanic potentiation Learn. Mem., January 29, 2009; 16(2): 129 - 141. [Abstract] [Full Text] [PDF]
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F/F0 (C). Note that the transient calcium concentration is elevated to a higher level in the presence of calpeptin (P < 0.05, n = 4).
