Naoto Saitoh

GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABAB receptor

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic γ-aminobutyric acid type B receptors (GABAB receptors) or metabotropic glutamate receptors inhibits presynaptic P/Q-type Ca2+ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein βγ subunits (Gβγ) directly into the calyceal nerve terminal through whole-cell patch pipettes. Gβγ slowed the activation of presynaptic Ca2+ currents (IpCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABAB receptor agonist. The effects of both Gβγ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, Gβγ partially occluded the inhibitory effect of baclofen on IpCa. In contrast, guanosine 5′-O-(3-thiotriphosphate)-bound Goα loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins Go, but not the Gi, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of Go βγ subunits with presynaptic Ca2+ channels.

Mechanisms underlying developmental speeding in AMPA-EPSC decay time at the calyx of Held

The time course of synaptic conductance is important in temporal precision of information processing in the neuronal network. The AMPA receptor (AMPAR)-mediated EPSCs at the calyx of Held become faster in decay time as animals mature. To clarify how desensitization and deactivation of AMPARs contribute to developmental speeding of EPSCs, we compared the decay time of quantal EPSCs (qEPSCs) with the deactivation and desensitization times of AMPAR currents induced in excised patches by fast glutamate application (AMPA patch currents). Both the deactivation and desensitization times of AMPA patch currents became markedly faster from postnatal day 7 (P7) to P14 and changed little thereafter. In individual neurons, throughout development (P7-P21), the time constants of deactivation and fast desensitization in AMPA patch currents were similar to each other and close to the qEPSC decay time constant. Cyclothiazide (CTZ) abolished the fast desensitization, prolonged deactivation of AMPA patch currents, and slowed the decay time of EPSCs. The effects of CTZ on AMPA patch currents were unchanged throughout development, whereas its effect on EPSCs became weaker as animals matured. In single-cell reverse transcription-PCR analysis, glutamate receptor subunit 4 (GluR4) flop increased from P7 to P14 and changed little thereafter. At P7, the GluR4 flop abundance had an inverse correlation with the qEPSC decay time. These results together suggest that both desensitization and deactivation of AMPARs are involved in the EPSC decay time, but the contribution of desensitization decreases during postnatal development at the calyx of Held.

Adenosine A1 receptor‐mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5‐ to 7‐day‐old (P5–7) rats, adenosine, or the type 1 adenosine (A1) receptor agonist N6‐cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A1 receptor antagonist 8‐cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca2+ currents (IpCa) recorded directly from calyceal terminals, but had no effect on presynaptic K+ currents. When EPSCs were evoked by IpCa during simultaneous pre‐ and postsynaptic recordings, the magnitude of the adenosine‐induced inhibition of IpCa fully explained that of EPSCs, suggesting that the presynaptic Ca2+ channel is the main target of A1 receptors. Whereas the N‐type Ca2+ channel blocker ω‐conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine‐induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A1 receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A1 receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca2+ channels.

Developmental shift to a mechanism of synaptic vesicle endocytosis requiring nanodomain Ca2

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ coordinates these processes remains unclear, particularly at mature synapses. Using membrane capacitance measurements from calyx of Held nerve terminals in rats, we found that vesicle endocytosis is initiated primarily in Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-dependent endocytosis required calmodulin and calcineurin activation at immature synapses, but not at more mature synapses. Similarly, GTP-independent endocytosis, which occurred after extensive exocytosis at immature synapses, became negligible after maturation. We propose that nanodomain Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse.

Activation of the epsilon isoform of protein kinase C in the mammalian nerve terminal

Activation of protein kinase C (PKC) by phorbol ester facilitates hormonal secretion and transmitter release, and phorbol ester-induced synaptic potentiation (PESP) is a model for presynaptic facilitation. A variety of PKC isoforms are expressed in the central nervous system, but the isoform involved in the PESP has not been identified. To address this question, we have applied immunocytochemical and electrophysiological techniques to the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of rat auditory brainstem. Western blot analysis indicated that both the Ca2+-dependent “conventional” PKC and Ca2+-independent “novel” PKC isoforms are expressed in the MNTB. Denervation of afferent fibers followed by organotypic culture, however, selectively decreased “novel” ɛPKC isoform expressed in this region. The afferent calyx terminal was clearly labeled with the ɛPKC immunofluorescence. On stimulation with phorbol ester, presynaptic ɛPKC underwent autophosphorylation and unidirectional translocation toward the synaptic side. Chelating presynaptic Ca2+, by using membrane permeable EGTA analogue or high concentration of EGTA directly loaded into calyceal terminals, had only a minor attenuating effect on the PESP. We conclude that the Ca2+-independent ɛPKC isoform mediates the PESP at this mammalian central nervous system synapse.

Involvement of AMPA receptor desensitization in short‐term synaptic depression at the calyx of Held in developing rats

Paired‐pulse facilitation (PPF) and depression (PPD) are forms of short‐term plasticity that are generally thought to reflect changes in transmitter release probability. However, desensitization of postsynaptic AMPA receptors (AMPARs) significantly contributes to PPD at many glutamatergic synapses. To clarify the involvement of AMPAR desensitization in synaptic PPD, we compared PPD with AMPAR desensitization, induced by paired‐pulse glutamate application in patches excised from postsynaptic cells at the calyx of Held synapse of developing rats. We found that AMPAR desensitization contributed significantly to PPD before the onset of hearing (P10–12), but that its contribution became negligible after hearing onset. During postnatal development (P7–21) the recovery of AMPARs from desensitization became faster. Concomitantly, glutamate sensitivity of AMPAR desensitization declined. Single‐cell reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated a developmental decline of GluR1 expression that correlated with speeding of the recovery of AMPARs from desensitization. Transmitter release probability declined during the second postnatal week (P7–14). Manipulation of the extracellular Ca2+/Mg2+ ratio, to match release probability at P7–8 and P13–15 synapses, revealed that the release probability is also an important factor determining the involvement of AMPAR desensitization in PPD. We conclude that the extent of involvement of AMPAR desensitization in short‐term synaptic depression is determined by both pre‐ and postsynaptic mechanisms.

Developmental changes in calcium/calmodulin-dependent inactivation of calcium currents at the rat calyx of Held

Ca2+‐binding to calmodulin (CaM) causes facilitation and/or inactivation of recombinant Ca2+ channels. At the rat calyx of Held, before hearing onset, presynaptic Ca2+ currents (IpCa) undergo Ca2+/CaM‐dependent inactivation during repetitive activation at around 1 Hz, implying that this may be a major cause of short‐term synaptic depression. However, it remains open whether the Ca2+/CaM‐dependent inactivation of IpCa persists in more mature animals. To address this question, we tested the effect of CaM inhibitors on the activity‐dependent modulation of IpCa in calyces, before (postnatal day (P) 7–9) and after (P13–15) hearing onset. Our results indicate that the CaM‐dependent IpCa inactivation during low‐frequency stimulation, and the ensuing synaptic depression, occur only at calyces in the prehearing period. However, CaM immunoreactivity in P8 and P14 calyces was equally strong. Even at P13–15, high frequency stimulation (200–500 Hz) could induce IpCa inactivation, which was attenuated by EGTA (10 mm) or a CaM inhibitor peptide loaded into the terminal. Furthermore, the CaM inhibitor peptide attenuated a transient facilitation of IpCa preceding inactivation observed at 500 Hz stimulation, whereas it had no effect on sustained IpCa facilitations during trains of 50–200 Hz stimulation. These results suggest that the Ca2+/CaM‐dependent IpCa modulation requires a high intraterminal Ca2+ concentration, which can be attained at immature calyces during low frequency stimulation, but only during unusually high frequency stimulation at calyceal terminals in the posthearing period.

Involvement of Ca2+ Channel Synprint Site in Synaptic Vesicle Endocytosis

The synaptic protein interaction (synprint) site of the voltage-gated Ca2+ channel (VGCC) α1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the μ subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2–synprint interaction occurred over a wide range of Ca2+ concentrations but was inhibited at high Ca2+ concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca2+-dependent interactions with AP-2 and Syt.

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli FORMATION, PREPARATION, AND UTILIZATION OF GIANT VACUOLE-LIKE STRUCTURES CONSISTING OF EVERTED CYTOPLASMIC MEMBRANE

Formation of giant protoplasts from normalEscherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H+because of respiration and to ATP hydrolysis by the FoF1-ATPase. Such current was inhibited by inhibitors of the respiratory chain or FoF1-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.

Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts ☆

Expansion mutation of CTG-repeat motifs within myotonin protein kinase (MtPK) gene is responsible for pathological changes in myotonic dystrophy (DM). To explore its pathological role in skeletal muscle, a full-length human MtPK cDNA was transfected into rat L6 myogenic cell line. Recombinantly expressed human MtPK protein in L6 cell line has a predicted molecular mass of 70 kDa. We have raised a polyclonal antibody against a synthetic peptide in the deduced sequence of the C-terminal portion of MtPK. MtPK in L6 cell is localized to perinuclear region, that resembles with sarcoplasmic reticulum. The MtPK-transfected myoblast cells established in this study will allow us to elucidate the molecular pathomechanism of muscle manifestations in DM.