syntaxin 1A in cultured rat hippocampal neurons

Neuroscience Letters 329 (2002) 273–276 www.elsevier.com/locate/neulet

Reduction of neurotransmitter release by the exogenous H3 domain peptide of HPC-1/syntaxin 1A in cultured rat hippocampal neurons Tatsuya Mishima*, Tomonori Fujiwara, Kimio Akagawa Department of Physiology, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan Received 21 May 2002; received in revised form 6 June 2002; accepted 11 June 2002

Abstract The membrane protein HPC-1/syntaxin 1A plays a key role in synaptic vesicle exocytosis in the presynaptic terminal. In particular, the H3 domain of HPC-1/syntaxin 1A participates in several protein–protein interactions that regulate neurotransmitter release. To investigate H3 domain function in neurotransmitter release, we used paired whole-cell patch clamping to record the evoked inhibitory postsynaptic currents in cultured hippocampal neurons. Introducing H3 domain peptide into the presynaptic neuron with a patch electrode depressed neurotransmitter release in a stimulation-frequency-dependent manner. Recovery from synaptic vesicle depletion induced by tetanic stimulation was significantly slowed by exogenous H3 domain peptide. These results suggest that the H3 domain peptide reduces neurotransmitter release by retarding the refilling of readily releasable vesicles. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: HPC-1/syntaxin 1A; SNAP-receptor; Inhibitory synaptic transmission; Exocytosis; Synaptic depletion; Hippocampal neuron

The exocytosis of neurotransmitters is a key event in neuronal cells. The process involves several steps: (1), docking, the initial contact between plasma membrane and synaptic vesicles; (2), priming, the maturation process that confers responsiveness to the initial Ca 21 influx; and (3), fusion of the synaptic vesicles with the plasma membrane [18]. HPC-1/syntaxin 1A is a plasma membrane protein [11] known as a SNARE (SNAP-receptor; soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor) that is indispensable for synaptic exocytosis. It binds to numerous proteins, including Ca 21 channels [1,17], complexin [16] and munc18 [9], and it binds synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptobrevin to form a SNARE complex [3,5,7]. Much of the binding has been mapped to the C-terminal H3 domain of HPC-1/syntaxin [4,14,20]. Although it has been reported that exogenous H3 fragment interrupts the interactions between endogenous HPC-1/syntaxin 1A and other proteins and inhibits transmitter release in squid giant synapse [15] and pheochromocytoma-12 (PC12) cells [8], the molecular mechanisms underlying this suppressive action are not yet clear. In this paper, we investigated the mechanisms of * Corresponding author. Tel.: 181-422-47-4801; fax: 181-42247-4801. E-mail address: [email protected] (T. Mishima).

recombinant H3 domain peptide inhibition of neurotransmitter release in mammalian central synapses. A low-density primary culture of hippocampal neurons was prepared from one-day-old neonatal rats. The neurons were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum, penicillin (50 IU/ml) and streptomycin (50 mg/ml), and plated on glial feeders on coverslips at a density of 2000–3000 cells/cm 2. After two days, one-third of the medium was replaced with serum-free, B27-supplemented (Gibco) DMEM. Cultures were used for electrophysiological recordings 10–21 days after plating. Paired whole-cell recordings were obtained from two hippocampal neurons whose soma lay within 100 mm. Patch clamp electrodes (4–6 MV) were fabricated from thin-walled borosilicate glass (G150TF-4, Warner Instruments) on a Flaming-Brown P-87 puller (Sutter Instruments). The presynaptic electrode contained 132 mM Kgluconate, 10 mM KCl, 5 mM N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (HEPES), 5 mM EGTA, 0.5 mM CaCl2, 2 mM MgCl2, 4 mM MgATP, 0.3 mM NaGTP, and 0.2 mM leupeptin, and the pH was adjusted to 7.3 with KOH. The postsynaptic electrode contained 90 mM Csgluconate, 20 mM KCl, 10 mM tetraethylammonium, 5 mM QX-314, 5 mM HEPES, 10 mM EGTA, 1 mM CaCl2, 2 mM MgCl2, 4 mM MgATP, 0.3 mM NaGTP,

0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 66 2- 6

274

T. Mishima et al. / Neuroscience Letters 329 (2002) 273–276

20 mM phosphocreatine, and 0.2 mM leupeptin, and the pH was adjusted to 7.3 with CsOH. Currents were collected through two amplifiers (EPC-9 for the postsynaptic neuron, HEKA and CEZ-2300 for the presynaptic neuron, Nihon Koden), and low-pass filtered at 2 kHz. Data were stored on DAT (RD-120, TEAC) and later digitized at 10 kHz and analyzed using Power Lab (AD Instruments). Series resistance was monitored continuously throughout all experiments by measuring the capacitative current response to a 5-mV voltage step, and was compensated 60%. If resistance changed by more than 10%, the experiment was discarded. The extracellular solution contained 126 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4. During recordings, 10 mM 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) and 50 mM DL-2Amino-5-phosphonovaleric Acid (APV) were added to the bath solution. A junction potential of 211 mV was corrected before sealing. All experiments were performed at room temperature (22–24 8C). Recombinant proteins were produced as described previously [8], except that purified proteins were desalted into solution for the presynaptic electrode. All data are presented as means ^ SEM. Levels of statistical significance were determined by paired Student’s t-tests. Mono-exponential curve fitting of data plot was done in IGOR (WaveMetrics). Both presynaptic and postsynaptic neurons were voltage clamped at a holding potential of 270 mV. Monosynaptic g-aminobutyric acidA receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked with a short latency (1–3 ms) when the presynaptic neuron was stimulated by stepping to 0 mV for 5 ms at 0.1–0.5 Hz. When whole-cell recordings were obtained with a normal intracellular solution, no obvious time-dependent rundown of IPSC amplitudes occurred with repetitive stimulation for at least 20 min. IPSC amplitude after 15 min of stimulation at 0.5 Hz was 106 ^ 12% of the mean of the first 1 min response. Introduction of the H3 domain peptide of HPC-1/syntaxin 1A (0.5 mg/ml) into the presynaptic neuron with a patch electrode caused a gradual decrease in the amplitude of evoked IPSC without affecting the time course of a single IPSC (Fig. 1). As a control, N-terminal fragment was introduced into the presynaptic neuron, but had no effect on evoked neurotransmission for over 15 min of observation. When the presynaptic neuron was stimulated at 0.5 Hz, the IPSC amplitude decreased to 55.2 ^ 4.5 and 81.9 ^ 7.4% at 15 min after the introduction of H3-peptide or N-peptide, respectively. A similar H3-peptide inhibitory effect was observed in (^)-a-amino-3-hydroxy-5-methylisoxazole-4propionic acid (AMPA) receptor-mediated evoked excitatory postsynaptic currents (data not shown). The H3 domain of HPC-1/Syntaxin 1A is involved in protein–protein interactions with N- and P/Q-type Ca 21 channels [1,14,17]. Binding of HPC-1/Syntaxin 1A to Ca 21 channels reduces current amplitude and modulates channel activation and inactivation [2,19]. To investigate whether the inhibitory effect of H3-peptide on neurotrans-

mission is related to the reduction of voltage-gated presynaptic Ca 21 currents, paired-pulse experiments were performed (Fig. 2). Pairs of stimuli separated by 200 ms were added twice to the presynaptic neuron immediately after, and at more than 15 min after establishing a wholecell recording with an electrode containing H3 domain peptide. Even after H3-peptide was adequately diffused in the presynaptic terminals, the paired-pulse ratio (0.80 ^ 0.07 to 0.77 ^ 0.14) and IPSC kinetics remained unchanged. We next characterized the influence of presynaptic neuron stimulus frequency to the inhibitory effect of H3peptide. Fig. 3 shows the time courses of transmitter release inhibition that were evoked by stimulation at 0.1, 0.2 and 0.5 Hz. At lower stimulus frequencies, the inhibitory effect of H3-peptide decreased, and at a stimulus frequency of 0.1 Hz, no reduction in transmitter release was observed during a 15-min experiment. At 15 min after recording, the amplitude of evoked IPSCs was 55.2 ^ 4.5% for 0.5 Hz, 69.6 ^ 14.3% for 0.2 Hz and 100.1 ^ 16.0% for 0.1 Hz.

Fig. 1. The H3-peptide of HPC-1/syntaxin 1A inhibits neurotransmitter release. (A) Representation of the recombinant fragments used. The three coiled-coil domains are indicated in black, and the transmembrane domain in gray. (B) IPSC amplitude was reduced by the introduction of H3-peptide, but not by N-peptide. (C) Average time course of IPSC amplitude depression during stimulation at 0.5 Hz. (Open circle) N-peptide infusion (n ¼ 9). (Filled circle) H3-peptide infusion (n ¼ 11). Each data point has been normalized to the average value recorded during the first minute after the onset of dual whole-cell recording. Data are statistically different (P , 0:05) from 90 to 120 s later.

T. Mishima et al. / Neuroscience Letters 329 (2002) 273–276

275

replenishment of readily releasable vesicles with a time constant of 20.7 s (Fig. 4). Our study showed the inhibitory effect of the recombinant H3 domain peptide of HPC-1/syntaxin 1A on neurotransmitter release in hippocampal neurons. The H3 domain interacts with a number of exocytosis-related proteins, including SNAP-25 [5], synaptobrevin [3], synaptotagmin [6], a-SNAP [10], munc18 [9], and Ca 21 channel [1,17]. The introduction of H3-peptide into the presynaptic terminals did not affect Ca 21-dependent release, suggesting that Ca 21 channels are not a target for H3-peptide. In the squid giant synapse, injected H3 domain peptide inhibited the binding of endogenous HPC-1/syntaxin 1A to SNAP-25, but had no effect on preformed binary or ternary SNARE complex [15]. In this study, the inhibitory effect of H3peptide on neurotransmission was stimulus-frequencydependent, and no suppression was observed for at least 15 min at 0.1 Hz (Fig. 3). This inhibitory effect was clearly observed when preformed SNARE complexes were thought to be consumed by tetanus stimulation (Fig. 4) [13]. Taken together, these results suggest that the reduction in neuro-

Fig. 2. The introduction of H3-peptide caused no change in the paired-pulse ratio. (A) Paired-pulse depressions of IPSC recorded from one cell at 1 and 17 min after H3-peptide infusion have been superimposed and normalized to IPSC1. Each trace is an average of five sweeps. (B) H3-peptide caused no significant change in the paired-pulse ratio, even after adequate diffusion (n ¼ 8).

When stimulus frequency was changed from 0.5 to 0.067 Hz at 15 min after recording, IPSC amplitude recovered to 84.7 ^ 12.2% within 15 s (n ¼ 4; data not shown). This stimulus-frequency-dependent suppression of transmitter release supports the idea that H3-peptide prevents endogenous HPC-1/syntaxin 1A from forming the ternary SNARE complex by binding newly-synthesized or disassembled SNARE proteins, and that it is incapable of interacting with preformed SNARE complex in readily releasable synaptic vesicles. We reported previously that suppression of transmitter release caused by internalized H3 fragment of HPC-1/syntaxin 1A in PC12 cells is facilitated when the preformed SNARE complex is disassembled in advance [8]. To further examine this idea, tetanus stimulation (20 Hz, 10 s) which appeared to exhaust pre-existing docked vesicles [12] was applied to the presynaptic neuron, and the recovery process was observed at 15 s intervals. This stimulation protocol was also chosen to increase the chance that H3-peptide interact with SNARE proteins by disassembling SNARE complex repeatedly. Although readily releasable synaptic vesicles recovered fully within 15 s of tetanus stimulation when the presynaptic electrode solution contained no H3-peptide, introduction of H3-peptide significantly depressed the onset of recovery and slowed the

Fig. 3. The stimulation-frequency-dependent effect of H3peptide. Each plot shows an average time course of IPSC amplitude depression during stimulation with 0.1 (n ¼ 5), 0.2 (n ¼ 6) and 0.5 Hz (n ¼ 11). Data points are plotted every 10 s. Each data point has been normalized to the average value during the first minute after the onset of dual whole-cell recording.

276

T. Mishima et al. / Neuroscience Letters 329 (2002) 273–276

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

Fig. 4. H3-peptide affects the recovery from synaptic vesicle depletion. (A) The introduction of H3-peptide slowed the recovery from synaptic vesicle depletion after tetanus stimulation (20 Hz, 10 s). (B) Average time course of recovery from tetanus stimulation. (Open circle) Normal intracellular solution was used as a control (n ¼ 12). (Filled circle) H3-peptide infusion (n ¼ 7). Each data point was normalized to the mean peak amplitude of four consecutive IPSCs before tetanus stimulation.

transmitter release is caused by delayed replenishment of readily releasable vesicles, and that H3-peptide alters the kinetics of SNARE complex assembly or vesicle recycling. This study was supported in part by a grant-in-aid from Promotion and Mutual Aid Cooperation for Private Schools of Japan, to K.A. [1] Bennett, M.K., Calakos, N. and Scheller, R.H., Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones, Science, 257 (1992) 255–259. [2] Bezprozvanny, I., Scheller, R.H. and Tsien, R.W., Functional impact of syntaxin on gating of N-type and Q-type calcium channels, Nature, 378 (1995) 623–626. [3] Calakos, N., Bennett, M.K., Peterson, K.E. and Scheller, R.H., Protein–protein interactions contributing to the specificity of intracellular vesicular trafficking, Science, 263 (1994) 1146–1149. [4] Canaves, J.M. and Montal, M., Assembly of a ternary complex by the predicted minimal coiled-coil-forming

[13]

[14]

[15]

[16]

[17]

[18] [19]

[20]

domains of syntaxin, SNAP-25, and synaptobrevin. A circular dichroism study, J. Biol. Chem., 273 (1998) 34214–34221. Chapman, E.R., An, S., Barton, N. and Jahn, R., SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils, J. Biol. Chem., 269 (1994) 27427–27432. Chapman, E.R., Hanson, P.I., An, S. and Jahn, R., Ca 21 regulates the interaction between synaptotagmin and syntaxin 1, J. Biol. Chem., 270 (1995) 23667–23671. Fasshauer, D., Eliason, W.K., Brunger, A.T. and Jahn, R., Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly, Biochemistry, 37 (1998) 10354–10362. Fujiwara, T., Yamamori, T. and Akagawa, K., Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein, Biochim. Biophys. Acta, 1539 (2001) 225–232. Hata, Y., Slaughter, C.A. and Sudhof, T.C., Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin, Nature, 366 (1993) 347–351. Haynes, L.P., Barnard, R.J., Morgan, A. and Burgoyne, R.D., Stimulation of NSF ATPase activity during t-SNARE priming, FEBS Lett., 436 (1998) 1–5. Inoue, A., Obata, K. and Akagawa, K., Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen, HPC-1, J. Biol. Chem., 267 (1992) 10613–10619. Kirischuk, S. and Grantyn, R., A readily releasable pool of single inhibitory boutons in culture, NeuroReport, 11 (2000) 3709–3713. Lonart, G. and Sudhof, T.C., Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles, J. Biol. Chem., 275 (2000) 27703–27707. Martin, F., Salinas, E., Vazquez, J., Soria, B. and Reig, J.A., Inhibition of insulin release by synthetic peptides shows that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca(2 1 )- but not for guanosine 5 0 -[gammathio]triphosphate-induced secretion, Biochem. J., 320 (1996) 201–205. O’Connor, V., Heuss, C., De Bello, W.M., Dresbach, T., Charlton, M.P., Hunt, J.H., Pellegrini, L.L., Hodel, A., Burger, M.M., Betz, H., Augustine, G.J. and Schafer, T., Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion, Proc. Natl. Acad. Sci. USA, 94 (1997) 12186–12191. Pabst, S., Hazzard, J.W., Antonin, W., Sudhof, T.C., Jahn, R., Rizo, J. and Fasshauer, D., Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions, J. Biol. Chem., 275 (2000) 19808–19818. Sheng, Z.H., Rettig, J., Takahashi, M. and Catterall, W.A., Identification of a syntaxin-binding site on N-type calcium channels, Neuron, 13 (1994) 1303–1313. Sudhof, T.C., The synaptic vesicle cycle: a cascade of protein–protein interactions, Nature, 375 (1995) 645–653. Wiser, O., Bennett, M.K. and Atlas, D., Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca 21 channels, EMBO J., 15 (1996) 4100–4110. Zhong, P., Chen, Y.A., Tam, D., Chung, D., Scheller, R.H. and Miljanich, G.P., An alpha-helical minimal binding domain within the H3 domain of syntaxin is required for SNAP-25 binding, Biochemistry, 36 (1997) 4317–4326.