Articles, Cellular/Molecular

Myosin Light Chain Kinase Accelerates Vesicle Endocytosis at the Calyx of Held Synapse

Hai-Yuan Yue and Jianhua Xu
Journal of Neuroscience 1 January 2014, 34 (1) 295-304; DOI: https://doi.org/10.1523/JNEUROSCI.3744-13.2014

Abstract

Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca2+ and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca2+/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca2+ channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca2+/calmodulin.

Introduction

At synaptic terminals, neuronal firing triggers vesicle exocytosis, which is followed by endocytosis to retrieve vesicle membrane. Endocytosis maintains the homeostasis of terminal membrane and recycles vesicles to sustain neurotransmission. Synaptic endocytosis is activity dependent and requires Ca2+ influx through voltage-gated Ca2+ channels on the plasma membrane (Sankaranarayanan and Ryan, 2001; Hosoi et al., 2009; Wu et al., 2009; Yamashita et al., 2010). In addition to synaptotagmin (Poskanzer et al., 2003; Yao et al., 2012), calmodulin is considered a primary Ca2+ sensor in endocytosis (Wu et al., 2009; Sun et al., 2010; Yamashita et al., 2010). Inhibition of calmodulin impairs different forms of endocytosis at the calyx of Held, including rapid endocytosis with a time constant of ∼2 s and slow clathrin-mediated endocytosis with a time constant of seconds to tens of seconds (Wu et al., 2009; Yamashita et al., 2010; Yao and Sakaba, 2012). In hippocampal neurons, knockdown of calmodulin expression decreases endocytosis rate (Sun et al., 2010). These observations further highlight calmodulin as a versatile Ca2+ sensor to regulate distinct synaptic functions including endocytosis, ion channel modulation (Xu and Wu, 2005; Dick et al., 2008; Catterall et al., 2013), vesicle replenishment (Sakaba and Neher, 2001), and synaptic plasticity (Lee et al., 2010). One downstream target of Ca2+/calmodulin is myosin light chain kinase (MLCK), which can phosphorylate the regulatory light chain of myosin (Nairn and Picciotto, 1994). Requirement of MLCK/myosin for presynaptic functions was first reported in sympathetic neurons (Mochida et al., 1994), followed by many studies demonstrating its regulation of synaptic strength and plasticity. For example, inhibitors of MLCK/myosin impair vesicle mobilization in hippocampal neurons (Ryan, 1999), disrupt high-frequency neuromuscular transmission (Polo-Parada et al., 2005; Seabrooke and Stewart, 2011), and prevent the increase of a readily releasable pool after tetanus stimulation in the immature calyx of Held (Lee et al., 2008). Furthermore, MLCK inhibitors increase the number of fast-releasing vesicles and facilitate basal neurotransmission in the calyx (Srinivasan et al., 2008; Lee et al., 2010). These effects indicate a regulatory role of MLCK in vesicle supply and neurotransmission. However, it remains unclear whether MLCK modulates synaptic vesicle endocytosis, which both contributes to vesicle supply and depends on Ca2+/calmodulin.

To determine the involvement of MLCK in synaptic endocytosis, we investigated effects of specific MLCK/myosin inhibitors on both slow and rapid forms of endocytosis at the calyx of Held, which can be readily resolved with the whole-cell membrane capacitance measurement. In our tests, MLCK inhibitory peptide 18 (MLCKip), ML-7, and wortmannin all decreased the kinetics of slow and rapid endocytosis and the efficiency of membrane recovery. Blocking myosin II activity with (S)-(−)-blebbistatin (blebbistatin) induced similar inhibition of endocytosis. The effects on endocytosis were not accompanied by changes in exocytosis and Ca2+ channel current. Our results thus suggest that MLCK/myosin facilitates endocytosis at synapses downstream of Ca2+ entry and exocytosis.

Materials and Methods

Slice preparation.

We prepared parasagittal brainstem slices containing the medial nucleus of the trapezoid body (MNTB; Xu and Wu, 2005) in accordance with guidelines of The Institutional Animal Care and Use Committee, Georgia Regents University. Brainstems were acquired from acutely decapitated 7- to 10-d-old Sprague Dawley rats of either sex, and chilled in ice-cold low Ca2+ artificial CSF (aCSF), which contained (in mm): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 3 MgCl2, 0.5 CaCl2, 25 glucose, 0.4 Na ascorbate, 3 myo-inositol, and 2 Na pyruvate, pH ∼7.4 when bubbled with 95% O2 and 5% CO2. Slices (180–200 μm thick) were sectioned using an automated VT1200S slicer (Leica Microsystems) and transferred into normal aCSF to recover at 37°C for 45 min before being left under room temperature (22–24°C). The normal aCSF was identical to the low Ca2+ aCSF except that it contained 1 mm MgCl2 and 2 mm CaCl2.

Electrophysiology.

Using the standard whole-cell patch-clamping technique (Xu and Wu, 2005; Xu et al., 2008), we measured membrane capacitance of the calyx of Held terminals in brainstem slices with either an EPC-10/2 amplifier or an EPC-9 amplifier controlled by the Patchmaster and Pulse program (HEKA), respectively. Capacitance was measured using a software lock-in function based on the Lindau-Neher method (Lindau and Neher, 1988), with a sinusoidal wave (60 mV peak-to-peak amplitude, 1000 Hz) being superimposed on a holding potential of −80 mV. The resulting capacitance data were sampled at 1 kHz without averaging, while membrane current was sampled at 20 kHz after an online Bessel filtering of 2.9 kHz. Ca2+ current, exocytosis, and endocytosis were evoked and analyzed during 4–12 min after establishing the whole-cell configuration. The bath solution contained the following (in mm): 105 NaCl, 20 TEA-Cl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 0.4 Na ascorbate, 3 myo-inositol, 2 Na pyruvate, 0.001 tetrodotoxin, and 0.1 3,4-diaminopyridine. The solution was 300–310 mOsm, and pH 7.4 when bubbled with 95% O2 and 5% CO2. The standard presynaptic pipette solution contained (in mm): 125 Cs-gluconate, 20 CsCl, 4 MgATP, 10 Na2-phosphocreatine, 0.3 GTP, 10 HEPES, 0.05 BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), 310–320 mOsm, pH adjusted to 7.2 with CsOH. As needed, the pipette solution was added with wortmannin (Abcam), MLCKip (sequence: RKKYKYRRK), dynamin inhibitory peptide (DYNip, sequence: QVPSRPNRAP), ML-7, blebbistatin, cyclosporin A (all from Tocris Bioscience), or calmodulin binding domain (CBD; sequence: LKKFNARRKLKGAILTTMLA; EMD Millipore Chemicals). The series resistance (6–15 MΩ) was compensated by 50–65% with a lag of 10 μs. For recording postsynaptic responses, the principal neuron of MNTB was voltage-clamped at −80 mV by a patch pipette filled with the following (in mm): 125 K-gluconate, 20 KCl, 4 MgATP, 10 Na2-phosphocreatine, 0.3 GTP, 10 HEPES, 0.5 EGTA, pH 7.2 (adjusted with KOH). The series resistance (5–15 MΩ) was compensated typically by 80% with a lag of 10 μs. The bath solution was added with 100 μm cyclothiazide, 1 μm strychnine chloride, and 10 μm bicuculline methiodide (all from Abcam) to block AMPA receptor desensitization, and postsynaptic currents mediated by glycine and GABA receptors, respectively. Recordings were made at room temperature (22–24°C).

Data analysis.

The amount of exocytosis (ΔCm) was measured as the difference between the averaged membrane capacitance (Cm) baseline before the stimulation and the peak of Cm transient. The initial rate of endocytosis, Rate_endo, was measured as the linear rate within the first 4 s of Cm decay following depolymerizaion for 20 ms (depol20ms) or the first 2 s of Cm decay following depol20ms×10 (Wu et al., 2009). To determine the efficiency of endocytosis, we also measured ΔCm25s or ΔCm45s, which was the difference between the Cm baseline and the Cm value at 25 or 45 s after termination of depol20ms or depol20ms×10. Time constant (τ) is provided in describing endocytosis kinetics in the control, but not in judging drug effects, because cases with extremely sluggish Cm decay weighed heavily in comparing τ between groups (Wu et al., 2009). Data are presented as mean ± SEM. The statistical test was Student's unpaired t test, with p < 0.05 indicating a significant difference.

Results

MLCKip impairs slow endocytosis

Endocytosis at the calyx depends on activity, with mild stimulation triggering slow clathrin-dependent, dynamin-dependent endocytosis and strong stimulation inducing an additional rapid form of endocytosis that depends on dynamin but probably not clathrin (Wu et al., 2005, 2009; Yamashita et al., 2005; Xu et al., 2008; Hosoi et al., 2009). Ca2+/calmodulin has been found to regulate both forms of endocytosis in immature calyces (Wu et al., 2009; Yamashita et al., 2010). Thus, we first investigated whether MLCK is involved in the classical slow endocytosis. We established the standard whole-cell patch-clamp configuration on the calyx terminals from 7- to 10-d-old rats, and monitored changes in Cm induced by membrane depolarization. At control calyces, a train of action potential-equivalent pulses (20 depolarization steps from −80 to 0 mV for 1 ms delivered every 9 ms, referred to as APeT below; Xu and Wu, 2005; Yamashita et al., 2005) triggered an abrupt Cm jump (ΔCm) of 322 ± 26 fF (n = 10), followed by monoexponential Cm decay with a τ of 14.7 ± 2.1 s toward the prestimulus level (Fig. 1Ai,B). Because Cm is proportional to the surface area of the terminals, ΔCm reflects exocytosis and correlates well with cumulative EPSCs, whereas Cm decay reflects endocytosis of membrane (Sun and Wu, 2001; Wu et al., 2005). The initial rate of Cm decay, Rate_endo, was 23 ± 1.8 fF/s in control (Fig. 1A,B). The net Cm increase at 25 s after the stimulation (ΔCm25s) was 56 ± 17 fF, reflecting nearly full recovery of the amount of exocytosed membrane. When the pipette solution contained 2 μm MLCKip, a water-soluble highly selective inhibitor of MLCK (Lukas et al., 1999), ΔCm slightly increased (366 ± 40 fF, n = 10, p = 0.37) while the following Cm decay became slower, resulting in a lower Rate_endo (9.2 ± 2.3 fF/s, p < 0.01) and more ΔCm25s (185 ± 34 fF, p < 0.01; Fig. 1Ai,B). To provide a positive control, we tested effects of DYNip, which has been shown to impair slow endocytosis at calyces (Hosoi et al., 2009) by interfering with dynamin functions. Consistent with the previous study, DYNip (100 μm) reduced Rate_endo (9.9 ± 3.6 fF/s, n = 6, p < 0.01) and raised ΔCm25s (144 ± 22 fF, p < 0.01). The similar effects of MLCKip and DYNip indicate that MLCKip is an effective inhibitor of the slow endocytosis induced by APeT.

Figure 1.
Figure 1.

MLCKip impairs slow endocytosis at the calyx. A, Sampled Cm recordings showing exocytosis and endocytosis induced by APeT (Ai) and depol20ms (Aii). The calyx terminals were dialyzed with the control pipette solution (Ctrl) or with the solution including MLCKip (2 μm), or DYNip (100 μm). B, ΔCm, Rate_endo, and ΔCm25s induced by APeT from control (n = 10), MLCKip (n = 10), and DYNip (n = 6), and induced by depol20ms from control (n = 10), MLCKip (n = 10), and DYNip (n = 8). **p < 0.01.

We continued to evaluate effects of MLCKip on slow endocytosis induced by a prolonged depolarization pulse, which is a widely used paradigm in the field (Wu et al., 2005; Renden and von Gersdorff, 2007; Hosoi et al., 2009; Sun et al., 2010; Yamashita et al., 2010). Consistent with previous studies, a depolarization pulse from −80 to 0 mV for 20 ms (referred to as depol20ms) triggered ΔCm of 437 ± 45 fF in control (n = 10) and the following Cm decay, which was fit with a monoexponential τ of 16.6 ± 1.6 s and an initial linear Rate_endo of 29.5 ± 4.0 fF/s (Fig. 1Aii,B). MLCKip did not change ΔCm (450 ± 35 fF, n = 10, p = 0.82), but prolonged Cm decay, leading to decreased Rate_endo (14.8 ± 2.8 fF/s, p < 0.01) and increased ΔCm25s (165 ± 18 fF vs 75 ± 21 fF in control, p < 0.01). DYNip similarly slowed down the Cm decay following depol20ms (Rate_endo, 11.8 ± 2.9 fF/s; ΔCm25s, 230 ± 30 fF; n = 8, p < 0.01 vs control; Fig. 1). Together, the results show that MLCKip can inhibit slow endocytosis induced by either APeT or depol20ms at calyces.

Our conclusion regarding inhibition of MLCKip on slow endocytosis is based on comparison of the kinetics of Cm decay following APeT and depol20ms. At the calyx, inhibition of MLCK induces mixed changes in synaptic transmission, including an increase in the number of fast-releasing vesicles as well as the frequency of miniature EPSCs (mEPSCs) at rest (Srinivasan et al., 2008). Also, compound fusion of vesicles is found following depolarization, leading to insertion of larger vesicle membrane into the plasma membrane during exocytosis (He et al., 2009; Xue and Wu, 2010). If inhibition of MLCK selectively promotes these changes during the asynchronous release immediately after depolarization, fusion of more and/or larger vesicles would slow down the initial Cm decay and confound our analysis of endocytosis kinetics. To address this concern, we studied effects of presynaptic dialysis of MLCKip on mEPSC by performing simultaneous patch-clamp recordings on both the calyx terminal and the postsynaptic principal neuron (Xu and Wu, 2005). The bath solution contained 100 μm cyclothiazide, which ensures accurate detection of asynchronous glutamate release after stimulation by blocking AMPA receptor desensitization. At control synapses (n = 6), the mEPSC frequency was 3.8 ± 0.8 Hz at rest and 10.3 ± 2.9 Hz (p = 0.06) within 4 s after APeT (Fig. 2A,C). At synapses with presynaptic dialysis of MLCKip (n = 7), the mEPSC frequency was 5.5 ± 1 Hz at rest and increased to 15 ± 3.2 Hz (p = 0.02) after APeT (Fig. 2B,C). In experiments in which APeT was replaced with depol20ms (Fig. 2D–F), the mEPSC frequency in control increased from 4.9 ± 1.2 Hz at rest to 12 ± 2.4 Hz (n = 6, p = 0.02) within 4 s after depol20ms, and in MLCKip from 5.2 ± 1.5 Hz at rest to 13.6 ± 3.8 Hz after depol20ms (n = 7, p = 0.08). Compared with control, MLCKip does not induce more fusion events before and after APeT or depol20ms (p = 0.23–0.76). We did not observe any unusual increase of mEPSC size in MLCKip either. The mean amplitude of mEPSCs over the first 4 s after stimulation fell within 28.2–30.6 pA in different conditions, showing no obvious effect of APeT, depol20ms, or MLCKip (p = 0.77–0.96, 6 control synapses and 7 MLCKip synapses; Fig. 2C,F). In summary, MLCKip does not affect either the number or the size of exocytosed vesicles following mild depolarization. Therefore, MLCKip slows down Cm decay following APeT or depol20ms by blocking endocytosis, not by modifying vesicle release after the stimulation.

Figure 2.
Figure 2.

MLCKip does not alter asynchronous release following mild stimulation. A, Sampled postsynaptic recordings from a control synapse under stimulation with APeT. The right two traces were expanded to show mEPSC events before and after APeT. B, Sampled postsynaptic recordings from a synapse with presynaptic dialysis of MLCKip (2 μm). Scales and arrangement of traces are identical with those in A. C, Frequency and amplitude of mEPSCs before and after APeT, which are mean and SEM of the averages in control (n = 6) and MLCKip synapses (n = 7). D, Sampled postsynaptic recordings from a control synapse under stimulation with depol20ms. The right two traces were expanded to show mEPSC events before and after depol20ms. E, Sampled postsynaptic recordings from a synapse with presynaptic dialysis of MLCKip. Scales and arrangement of traces are identical with those in D. F, Frequency and amplitude of mEPSCs before and after depol20ms in control (n = 6) and MLCKip (n = 7). Note that stimulation with APeT or depol20ms significantly increases the mEPSC frequency (*p < 0.05 when applicable). The mEPSC frequency in MLCKip is not significantly different (n.s.) from that in control.

Inhibitors of MLCK/myosin impair slow endocytosis

The inhibitory effects of MLCKip on slow Cm decay suggest that MLCK accelerates slow endocytosis. To verify this conclusion, we examined whether the slow endocytosis induced by depol20ms is affected by ML-7 and wortmannin, which are two additional inhibitors of MLCK widely used in studying neuronal functions (Polo-Parada et al., 2005; Tokuoka and Goda, 2006; Lee et al., 2008, 2010; Srinivasan et al., 2008). ML-7 competes with ATP for binding with MLCK (Saitoh et al., 1987), and wortmannin targets at or near the catalytic site of MLCK at a concentration ≥ 1 μm (Nakanishi et al., 1992). Because the final solutions of the blockers contained 0.1% DMSO, we used 0.1% DMSO in the pipette solution as their control. Consistent with previous studies (Xu et al., 2008; Wu et al., 2009; Yamashita et al., 2010), DMSO did not influence either exocytosis or endocytosis following depol20ms (Fig. 3A,B). Specifically, ΔCm reflecting exocytosis was 442 ± 36 fF (n = 10, p = 0.93), Rate_endo was 29.7 ± 3.2 fF/s (p = 0.96), and ΔCm25s was 73 ± 22 fF (p = 0.94; Fig. 3A,B). By contrast, including ML-7 (20 μm) in the pipette solution significantly prolonged Cm decay, resulting in decreased Rate_endo (12.6 ± 1.9 fF/s, n = 10, p < 0.01) and increased ΔCm25s (214 ± 22 fF, p < 0.01). Dialysis with wortmannin (10 μm) also reduced Rate_endo (7.9 ± 1.4 fF/s, n = 8, p < 0.01) and increased ΔCm25s (260 ± 58 fF, p < 0.01; Fig. 3A,B). Thus, all three MLCK blockers, MLCKip, ML-7, and wortmannin, consistently inhibited slow endocytosis.

Figure 3.
Figure 3.

ML-7, wortmannin, and blebbistatin inhibit slow endocytosis. A, Sampled membrane capacitance (Cm) recordings showing exocytosis and endocytosis induced by depol20ms from the calyx terminals dialyzed with pipette solution containing either DMSO (0.1%), ML-7 (20 μm), wortmannin (Wort, 10 μm), or blebbistatin (Bleb, 100 μm). B, ΔCm, Rate_endo and ΔCm25s induced by depol20ms from DMSO (n = 10), ML-7 (n = 10), wortmannin (n = 8) and blebbistatin (n = 10). **p < 0.01.

Activation of MLCK can phosphorylate the light chain of myosin. To determine whether myosin acts together with MLCK in regulating slow endocytosis, we examined effects of dialysis with blebbistatin, which selectively blocks the ATPase activity of myosin II (Kovacs et al., 2004) and has been shown to inhibit refilling of fast-releasing vesicles after tetanic stimulation at the calyx (Lee et al., 2008, 2010). Blebbistatin (100 μm) decreased Rate_endo after depol20ms to 9.3 ± 3.1fF/s (n = 10, p < 0.01) and increased ΔCm25s to 228 ± 32 fF (p < 0.01; Fig. 3A,B), suggesting that myosin II is involved in slow endocytosis. Based on the effects of three MLCK inhibitors (MLCKip, ML-7, and wortmannin) and a myosin II inhibitor (blebbistatin) on slow endocytosis, we conclude that MLCK activity facilitates the classical slow endocytosis. None of the blockers affected ΔCm (Figs. 1B, 3B), ruling out the possibility that their inhibition of slow endocytosis results from modulation of exocytosis.

MLCKip slows down the rapid endocytosis following intense stimulation

Having shown that MLCK accelerates slow endocytosis, we next investigated whether MLCK modulates a rapid form of endocytosis induced by intense activity (Wu et al., 2005). In control, a 10 Hz train of ten 20 ms depolarization pulses from −80 to 0 mV (referred to as depol20ms×10) evoked a ΔCm of 1266 ± 105 fF (n = 10) and a biexponential Cm decay with a rapid τ of 2.4 ± 0.4 s (median = 2.1 s, 34.1 ± 4.8% of the total amount) and a slow τ of 20.6 ± 2.1 s (median = 18.2 s; Fig. 4A,C). The Rate_endo measured within 2 s of Cm decay after depol20ms×10 was 177 ± 15 fF/s, reflecting the rate of membrane retrieval primarily through the rapid mechanism (Wu et al., 2005, 2009; Xu et al., 2013). Introducing MLCKip into the terminals decreased Rate_endo to 113 ± 24 fF/s (n = 10, p = 0.03; Fig. 4A,B), and prolonged both rapid and slow τ (median = 4.0 s and 54.1 s, respectively) without changing the contribution from rapid endocytosis (33.5 ± 5.5%), which resulted in larger ΔCm45s (332 ± 56 fF, p < 0.01) than that in control (101 ± 22 fF). Because rapid endocytosis is inhibited by dynamin blockers (Xu et al., 2008), we used the dynamin inhibitor, DYNip, as a positive control. DYNip decreased Rate_endo (106 ± 10 fF/s, n = 6, p < 0.01) and increased ΔCm45s (315 ± 46 fF, p < 0.01) to an extent similar to that of MLCKip (Fig. 4). Like other specific inhibitors of dynamin (Xu et al., 2008), DYNip increased both rapid and slow τ (median = 4.6 s and 66.5 s, respectively) without altering the contribution of rapid endocytosis (42.3 ± 5.8%, p = 0.31). These results indicate that MLCKip effectively inhibits the rapid Cm decay induced by depol20ms×10.

Figure 4.
Figure 4.

MLCKip slows down rapid endocytosis following intense stimulation. A, Sampled Cm recordings showing exocytosis and endocytosis induced by depol10×20ms from calyces dialyzed with the control, MLCKip (2 μm), or DYNip (100 μm) solution. B, ΔCm, Rate_endo and ΔCm5s induced by depol10×20ms from control (n = 10), MLCKip (n = 10), and DYNip (n = 6). *p < 0.05, **p < 0.01.

To determine whether MLCKip slows down rapid Cm decay by promoting asynchronous vesicle fusion or the size of fused vesicles after depol20ms×10, we measured mEPSCs from the postsynaptic neurons innervated by the calyx terminals under dialysis with MLCKip or control pipette solution. In control synapses (n = 6), the mEPSC frequency increased from 4.4 ± 0.7 Hz at rest to 31.4 ± 2 Hz (p < 0.01) during 3 s after depol20ms×10. The averaged mEPSC amplitude increased from 26 ± 1.5 pA to 29.6 ± 2.1 pA (p = 0.17) after the stimulation (Fig. 5A,C), which agrees with an increasing trend of mEPSC size after prolonged repetitive depolarization (He et al., 2009; Xue and Wu, 2010; Fioravante et al., 2011). MLCKip did not further increase the amplitude or frequency elevation in mEPSC events (Fig. 5B,C). Therefore, MLCKip slows down the rapid Cm decay after depol20ms×10 by inhibition of rapid membrane retrieval, not by facilitation of asynchronous vesicle fusion.

Figure 5.
Figure 5.

MLCKip does not alter asynchronous release following intense stimulation. A, Sampled postsynaptic recordings from a control synapse under stimulation with depol20ms×10. The right two traces were expanded to show mEPSC events before and after the stimulation. As shown in the inset, large grooves in postsynaptic currents are composed of multiple narrowly separated mEPSC events, which appear more frequently after depol20ms×10. B, Sampled postsynaptic recordings from a synapse with presynaptic dialysis of MLCKip (2 μm). Scales and arrangement of traces are identical with those in A. C, Frequency and amplitude of mEPSCs before and after depol20ms×10. The mEPSC frequency drastically increases after depol20ms×10 (**p < 0.01) in both control (n = 6) and MLCKip (n = 6), while the amplitude increases less significantly. MLCKip does not induce further changes.

Inhibitors of MLCK/myosin slow down rapid endocytosis

To verify the involvement of MLCK in rapid endocytosis, we further examined effects of ML-7, wortmannin, and blebbistatin (Fig. 6). In calyces dialyzed with DMSO (0.1%), depol20ms×10 induced both rapid and slow components which were similar to control, with a Rate_endo of 185.9 ± 25.4 fF/s (n = 10, p = 0.78) and ΔCm45s of 143 ± 33 fF (p = 0.31). ML-7 (20 μm) reduced Rate_endo to 112.6 ± 13.5 fF/s (n = 10, p = 0.02) and increased ΔCm45s of 335 ± 73 fF (p = 0.03). Similar effects were observed for wortmannin (10 μm) and blebbistatin (100 μm; Fig. 6A,B). As determined by the biexponential fitting of Cm decay, ML-7, wortmannin, and blebbistatin all increased rapid τ (median = 3.6, 3.4, and 3.7 s, respectively, vs 2.0 s in DMSO) and slow τ (median = 53.8, 69.5, and 50.0 s, respectively, vs 26.3 s in DMSO) without changing their proportions (rapid component: 34.4 ± 4.1%, 37.6 ± 8.7%, and 30.5 ± 3.9%, respectively, vs 34.2 ± 3.9% in DMSO). Therefore, the four blockers of MLCK/myosin decreased not only the rate of slow endocytosis induced by APeT or depol20ms but also the rate of rapid membrane retrieval following depol20ms×10. Meanwhile, ΔCm following depol20ms×10 was slightly smaller in MLCK/myosin blockers, which is probably caused by slower clearance of fused membrane from active zones due to impairment of rapid endocytosis (Hosoi et al., 2009; Wu et al., 2009; Hua et al., 2013). The mild reduction of ΔCm should not underlie the impairment of endocytosis.

Figure 6.
Figure 6.

ML-7, wortmannin, and blebbistatin slow down rapid endocytosis following intense stimulation. A, Sampled Cm recordings showing exocytosis and endocytosis induced by depol20ms×10 from calyces dialyzed with DMSO (0.1%), ML-7 (20 μm), wortmannin (10 μm), or blebbistatin (100 μm). B, ΔCm, Rate_endo and ΔCm45s induced by depol20ms×10 from DMSO (n = 10), ML-7 (n = 10), wortmannin (n = 8), and blebbistatin (n = 10).

MLCK/myosin functions downstream of Ca2+ influx

At the calyx, the kinetics of endocytosis induced by membrane depolarization depends on Ca2+ entry via voltage-gated Ca2+ channels (Xue et al., 2012). Among the inhibitors we used, ML-7 has been found to reduce Ca2+ channel current in cultured hippocampal neurons (Tokuoka and Goda, 2006). To exclude interference of possible reduction of Ca2+ entry in our experimental conditions, we measured Ca2+ influx induced by depol20ms and depol20ms×10. In control, depol20ms induced Ca2+ current with a peak amplitude (ICapeak) of 2.0 ± 0.2 nA (n = 10) and a current charge (QCa) of 32.9 ± 2.5 pC (Fig. 7B). For the treatments of DMSO and blockers of MLCK/myosin, ICapeak fell between 2 and 2.1 nA and QCa between 32.3 and 35.5 pC, showing no difference from control (p = 0.77–0.93 and 0.82–0.92, respectively). Depol20ms×10, which repeats depol20ms for 10 times at an interval of 80 ms, induced a total QCa of 227.2 ± 19.2 pC (n = 10) in control (Fig. 7D). Depol20ms×10 evoked similar total QCa in DMSO and the blockers (QCa = 212.4–246.1 pC, n = 8–10, p = 0.26–0.92). MLCKip, ML-7, wortmannin, and blebbistatin did not change ICa (channel current) or QCa under depol20ms and depol20ms×10, which is consistent with a previous study using brief depolarization pulses (Srinivasan et al., 2008). Therefore, the inhibitors of MLCK/myosin II impair slow and rapid endocytosis by acting downstream of Ca2+ influx.

Figure 7.
Figure 7.

Inhibition of MLCK/myosin II does not affect voltage-gated Ca2+ channel current. A, Sampled Ca2+ channel current (ICa) evoked by depol20ms in a control calyx. B, Peak amplitude (ICapeak) and total charge (QCa) of ICa evoked by depol20ms from calyces dialyzed with different pipette solutions (control, n = 10; MLCKip, n = 10; DMSO, n = 10; ML-7, n = 10; wortmannin, n = 8; blebbistatin, n = 10). C, Sampled ICa evoked by depol20ms×10 in a control calyx. D, Total charge (QCa) of ICa evoked by depol20ms×10 from calyces dialyzed with different pipette solutions (control, n = 10; MLCKip, n = 10; DMSO, n = 10; ML-7, n = 10; wortmannin, n = 8; blebbistatin, n = 10).

Simultaneous inhibition of MLCK and calmodulin does not induce synergistic inhibition of endocytosis

Downstream of Ca2+ influx, calmodulin has been shown to regulate endocytosis in the calyx of Held and hippocampal boutons (Wu et al., 2009; Sun et al., 2010; Yamashita et al., 2010; Yao and Sakaba, 2012). To test whether MLCK and calmodulin regulate endocytosis along the same pathway, we investigated whether combining a calmodulin inhibitor, CBD, and MLCKip could produce synergistic inhibition of endocytosis. Consistent with the previous studies (Wu et al., 2009; Yamashita et al., 2010), CBD at 500 μm significantly inhibited both slow and rapid endocytosis (Fig. 8). Specifically, CBD reduced Rate_endo following depol20ms to 10.2 ± 2.8 fF/s (n = 8, p < 0.01 vs control in Fig. 1) and increased ΔCm25s to 268 ± 35 fF (p < 0.01; Fig. 8). CBD increased ΔCm25s more than MLCKip (165 ± 18 fF in Fig. 1, p = 0.01), but decreased Rate_endo to a similar extent (p = 0.26), showing mildly stronger inhibition of endocytosis. Combining CBD and MLCKip in the pipette solution resulted in a Rate_endo of 8.4 ± 2.2 fF/s (n = 8, p < 0.01 vs control) and a ΔCm25s of 295 ± 47 fF (p < 0.01). The changes are similar to those in CBD alone (p = 0.64 and 0.66, respectively). Combining CBD and MLCKip thus does not generate synergistic effects on slow endocytosis.

Figure 8.
Figure 8.

Simultaneous inhibition of calmodulin and MLCK does not induce additive inhibition of endocytosis. A, Sampled Cm changes induced by depol20ms from calyces dialyzed with the pipette solution containing CBD (500 μm) along with or without MLCKip (2 μm). B, Averaged Cm responses induced by depol20ms in control (n = 10), CBD (n = 8), MLCKip (n = 10), and CBD with MLCKip (n = 8), with the error bars indicating SEM. Individual Cm traces were normalized by ΔCm before averaging. Data of control and MLCKip are from the same tests as shown in Figure 1. C, Comparison of ΔCm, Rate_endo, and ΔCm25s induced by depol20ms. D, Sampled Cm changes induced by depol20ms×10 from calyces dialyzed with CBD and/or MLCKip. E, Averaged Cm responses induced by depol20ms×10 in control (n = 10), CBD (n = 7), MLCKip (n = 10), and CBD with MLCKip (n = 6), which are indicated by the same colors as in B. Data of control and MLCKip are from the same tests as shown in Figure 4. Individual Cm traces were normalized by ΔCm before averaging. F, Comparison of ΔCm, Rate_endo and ΔCm25s induced by depol20ms×10.

Similar comparison was done on rapid endocytosis (Fig. 8C,D). CBD decreased the rapid Rate_endo following depol20ms×10 to 80.1 ± 11.2 fF/s (n = 7, p < 0.01 vs control), and raised ΔCm45s to 342 ± 60 fF (p < 0.01). The effects are indistinguishable from those of MLCKip (p = 0.29 and 0.91, respectively). The combination of CBD and MLCKip reduced Rate_endo to 76.3 ± 14.2 fF/s (n = 6) and increased ΔCm25s to 467 ± 147 fF, which are similar to those in CBD (p = 0.83 and 0.42, respectively) and MLCKip (p = 0.28 and 0.33, respectively). It is noteworthy that neither individual blockers nor combination of MLCKip and CBD completely abolishes endocytosis, which is seen instead for the rapid Ca2+ chelator, BAPTA (Hosoi et al., 2009; Wu et al., 2009; Yamashita et al., 2010). Thus, although the depolarization-evoked endocytosis in the calyx requires Ca2+, our pharmacological data do not exclude the possible existence of a Ca2+ sensor other than calmodulin (such as synaptotagmin). Nevertheless, combining MLCKip and CBD did not induce synergistic inhibition on slow endocytosis or rapid endocytosis, suggesting that MLCK and calmodulin function through the same pathway to affect endocytosis.

Discussion

We have observed at the calyx of Held from P7–P10 rats that acutely inhibiting MLCK/myosin with MLCKip, ML-7, wortmannin, and blebbistatin impaired slow endocytosis following APeT and depol20ms (Figs. 1, 3), and rapid endocytosis following depol20ms×10 (Figs. 4, 6). We conclude that under both mild and intense activity, MLCK/myosin accelerates endocytosis in this mammalian central synapse.

A role of MLCK/myosin in synaptic vesicle endocytosis

In non-neuronal cells, MLCK/myosin has been reported to regulate different forms of endocytosis, including caveolar endocytosis in intestinal epithelia (Schwarz et al., 2007; Marchiando et al., 2010), phagocytosis and pseudopod formation in polymorphonuclear leukocytes (Mansfield et al., 2000), and receptor-mediated phagocytosis and macropinocytosis in macrophages (Araki et al., 2003). In neurons, a role of MLCK/myosin in endocytosis has remained in doubt. An early study on hippocampal neurons found that inhibition of MLCK/myosin with ML-9 or a nonspecific myosin inhibitor, butanedione monoxime, can attenuate the destaining but not the uptake of FM1-43 (Ryan, 1999). The study suggests that MLCK is not involved in endocytosis under investigation, but does not exclude its participation under different conditions. In the present study, using capacitance measurement to monitor endocytosis with a temporal resolution as high as milliseconds, we found that four blockers of MLCK/myosin all slowed down endocytosis after either mild or intense stimulation, causing impairment similar to or slightly weaker than that of the dynamin inhibitor DYNip (Figs. 1, 3, 4, 6) or the calmodulin inhibitor CBD (Fig. 8), which are recognized potent inhibitors of endocytosis at the calyx (Hosoi et al., 2009; Wu et al., 2009; Yamashita et al., 2010). Our data thus indicate that, like in non-neuronal cells, MLCK/myosin accelerates vesicle endocytosis in the calyx synapse.

Given that MLCK and myosin are widely expressed in the nervous system, facilitation of endocytosis by MLCK/myosin may not be limited to the calyx. In the mouse motor nerve terminals, MLCK/myosin sustains high-frequency neurotransmission by activating a rapid reuse mode of vesicle cycling (Maeno-Hikichi et al., 2011). Whether this rapid reuse results from accelerated endocytosis is an interesting question to explore. Like most neurophysiology studies, the current paper has relied on using selective blockers to disrupt MLCK/myosin functions. Since the global deletion of MLCK, myosin IIA, or myosin IIB is lethal at birth, future studies using cultured embryonic neurons and conditional knock-out animals will be necessary to study the role of MLCK/myosin in endocytosis on the basis of specifically controlled expression of MLCK and myosin. Indeed, a new study reports that knock-out of myosin IIB decreases activity-dependent uptake of FM1-43 and horseradish peroxidase in cultured hippocampal neurons (Chandrasekar et al., 2013), suggesting that MLCK/myosin positively modulates endocytosis across different synapses.

Possible mechanisms underlying the facilitation of endocytosis by MLCK/myosin

Inhibitors of MLCK/myosin reduced endocytosis kinetics without affecting Ca2+ current (Fig. 7) and ΔCm following depolarization (Figs. 1, 3, 4, 6), indicating that MLCK functions downstream of Ca2+ and vesicle fusion in regulating endocytosis. MLCK is typically activated by Ca2+/calmodulin and can phosphorylate the regulatory light chain of myosin, which triggers interaction between myosin and actin. Although MLCK may also be directly or indirectly activated by other molecules such as the neural cell adhesion molecule (Polo-Parada et al., 2005), protein kinase C (Maeno-Hikichi et al., 2011), Rho/Rho-kinase (Garcia et al., 1999), and p21-activated kinases (Sanders et al., 1999), its modulation of synaptic endocytosis most likely involves Ca2+/calmodulin that has been indicated to regulate endocytosis in neurons (Wu et al., 2009; Sun et al., 2010; Yamashita et al., 2010; Yao and Sakaba, 2012) and neuroendocrine cells (Artalejo et al., 1996). Calmodulin inhibition at the calyx impairs distinct forms of endocytosis induced by different stimulation paradigms (Wu et al., 2009; Yamashita et al., 2010; Yao and Sakaba, 2012), including slow and rapid endocytosis (Wu et al., 2009; Yamashita et al., 2010). Consistently, we observed a calmodulin inhibitor, CBD, significantly reduced slow and rapid endocytosis (Fig. 8). Combining CBD and MLCKip did not induce further inhibition of endocytosis (Fig. 8), suggesting that both inhibitors target the same mechanism of endocytosis. Therefore, we think that MLCK accelerates endocytosis downstream of Ca2+/calmodulin.

A phosphatase downstream of Ca2+/calmodulin, calcineurin, has been indicated to regulate endocytosis in the calyx and hippocampal boutons (Sun et al., 2010; Yamashita et al., 2010), probably by phosphorylation of dynamin (Liu et al., 1994). We confirmed the involvement of calcineurin in rapid and slow endocytosis by reproducing inhibitory effects of a calcineurin blocker, cyclosporin A (data not shown). Our new finding thus suggests that calmodulin can activate different pathways to regulate endocytosis. Activation of MLCK can phosphorylate myosin, which interacts with actin. In our experiments, blocking myosin II with blebbistatin impaired both slow and rapid endocytosis (Figs. 3, 6). Blebbistatin and deletion of myosin IIB also inhibit endocytosis in hippocampal synapses (Chandrasekar et al., 2013). Although a role of actin in endocytosis is not yet established at mammalian central synapses (Sankaranarayanan et al., 2003), it has been demonstrated in other synapses and non-neuronal cells (Brodin et al., 2000; Merrifield et al., 2005; Girao et al., 2008; Mooren et al., 2012). In the lamprey synapses, actin-depolymerizing agents can inhibit clathrin-mediated endocytosis and arrest clathrin-coated pits on the plasma membrane (Shupliakov et al., 2002; Bourne et al., 2006). Inhibitors of actin cytoskeleton also impair clathrin-independent endocytosis in pancreatic β cells (He et al., 2008). Actin may assist in detaching vesicle membrane from the plasma membrane along with dynamin (Lee and De Camilli, 2002; Schafer, 2002; Gu et al., 2010). Like dynamin inhibition, disruption of actin polymerization delays the closure of fission pores to retrieve vesicle membrane in chromaffin cells, suggesting that actin indeed regulates the last step of endocytosis (Yao et al., 2013). Based on these results, we suggest that MLCK/myosin facilitates synaptic vesicle endocytosis by functioning between Ca2+/calmodulin and actin. The MLCK/myosin pathway and the calcineurin pathway may cooperatively regulate vesicle scission through actin and dynamin, respectively. Because the blockers caused similar partial inhibition of endocytosis, it is difficult to pharmacologically determine the relative contributions of these two pathways downstream of Ca2+/calmodulin. A future approach is to test inhibitors on synapses from animals lacking MLCK, myosin II, or calcineurin (subunits).

Significance of MLCK/myosin in synaptic transmission

The current study assigns acceleration of endocytosis as a new role for MLCK/myosin, which was first proposed to function in nerve terminals two decades ago (Mochida et al., 1994). Because endocytosis supports neurotransmission by removing fused vesicle membrane from release sites and by recycling vesicles for future exocytosis (Hosoi et al., 2009; Wu et al., 2009; Hua et al., 2013), an enhancement role in endocytosis predicts contribution of MLCK/myosin to vesicle cycling and synaptic strength during sustained activity, which is compatible with the reported functions of MLCK/myosin in synaptic physiology.

The current literature suggests that MLCK/myosin facilitates vesicle mobilization for release in nerve terminals. Inhibition of MLCK/myosin inhibits high-frequency neurotransmission in motor neurons (Maeno-Hikichi et al., 2011; Seabrooke and Stewart, 2011), and reduces vesicle trafficking and exocytosis in hippocampal neurons (Ryan, 1999), which was later suggested to result from nonspecific inhibition of Ca2+ current (Tokuoka and Goda, 2006). At the calyx, MLCK/myosin inhibitors decrease post-tetanic potentiation following high-frequency action potential firing, without changing Ca2+ current (Lee et al., 2008, 2010). The effect is attributed to impaired recruitment of fast-releasing vesicles, which depends on Ca2+/calmodulin (Sakaba and Neher, 2001) and actin (Sakaba and Neher, 2003). Because vesicle replenishment and endocytosis both require Ca2+/calmodulin and MLCK/myosin, one may wonder whether replenishment of fast-releasing vesicles depends on the speed of endocytosis. However, this possibility is excluded by the finding that inhibition of protein kinase A slows endocytosis but not vesicle replenishment (Yao and Sakaba, 2012). In addition, vesicles recruited for release are reported to come from a large recycling pool (Xue et al., 2013), instead of the pool of new vesicles generated from endocytosis (Wu and Wu, 2009). In light of these studies, it is possible that MLCK/myosin facilitates vesicle endocytosis and mobilizes vesicles for release independently.

Meanwhile, MLCK is found to negatively regulate basal neurotransmission. Blocking MLCK activity in the calyx increases the number of fast-releasing vesicles and facilitates the EPSCs evoked by the early action potentials during a train stimulus (Srinivasan et al., 2008; Lee et al., 2010). Similarly, MLCK blockers can antagonize the reduction of releasable vesicles caused by inhibition of Rho kinase in hypoglossal motor synapses (González-Forero et al., 2012). These observations suggest that constitutive activity of MLCK limits the number of vesicles acquiring high priority in fusion. Based on the seemingly paradoxical effects of MLCK inhibitors in vesicle cycling and basal release, we speculate that the constitutive activity of MLCK restricts consumption of vesicles in the readily releasable pool, and activation of MLCK by Ca2+/calmodulin accelerates both vesicle regeneration via endocytosis and vesicle replenishment from an existing pool. The different functions of MLCK help nerve terminals to maintain continuous supply of releasable vesicles in an activity-dependent manner. To summarize, the present study suggests that activation of MLCK/myosin accelerates the Ca2+/calmodulin-dependent vesicle endocytosis in synapses, which can contribute to sustaining the neurotransmission during repetitive activity.

Footnotes

  • This work has been supported by the start-up fund from Georgia Regents University. We thank Drs. Darrell Brann and Quan-Sheng Du for providing constructive comments on this manuscript.

  • Correspondence should be addressed to Jianhua Xu, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912. jxu1{at}gru.edu

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Myosin Light Chain Kinase Accelerates Vesicle Endocytosis at the Calyx of Held Synapse
Hai-Yuan Yue, Jianhua Xu
Journal of Neuroscience 1 January 2014, 34 (1) 295-304; DOI: 10.1523/JNEUROSCI.3744-13.2014
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