SNAP-25b的磷酸化調控分泌細胞的胞吐作用動態及發育中大鼠的視網膜波

SNAP-25b phosphorylation modulates the exocytotic kinetics in secretory cells and retinal waves in the developing rat retina

蕭愉恬(Yu-Tien Hsiao)

指導教授 ： 王致恬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.10101

摘要

SNARE複合體可調控囊泡融合。在三個SNARE蛋白質中，僅SNAP-25（SN25）可被蛋白質激酶A（protein kinase A，PKA）和蛋白質激酶C（protein kinase C，PKC）磷酸化，且磷酸化位置分別在SN25的T138和S187。以往的研究顯示，PKA或PKC磷酸化的SN25可藉由控制囊泡池（vesicle pool）的大小或補充囊泡池來調控囊泡的分泌量。然而，目前仍不清楚（1）SN25的磷酸化如何調控胞吐作用的動態;（2）此調控是否可以顯著影響大規模的網路活動。為了解決第一個問題，我們利用可測得單一囊泡釋放的氧化電流技術（single-event amperometry），在PC12細胞內研究SN25b的磷酸化對胞吐動態的影響，尤其是對融合孔（fusion pore）動態的影響。此研究中，我們使用兩種不同secretogogues在短時間內(約1分鐘)誘發鈣離子依賴性胞吐作用（Ca2+-dependent exocytosis），包括高濃度氯化鉀（KCl）、高濃度氯化鉀與福斯克林（Forskolin，一種腺苷酸環化酶的激活劑；FSK）的混合液（KCl FSK）。與對照組相比，KCl降低過量表現SN25b細胞的囊泡釋放速率，但KCl FSK更為顯著降低過量表現SN25b細胞的囊泡釋放速率，藉由免疫沉澱實驗顯示KCl FSK可使SN25b被PKA磷酸化，降低鈣離子依賴性胞吐作用的速率。此外， 和SN25b相比，過量表現SN25b突變株（SN25b-T138A或SN25b-S187A）的細胞，其囊泡釋放速率增加，顯示經由被PKA或PKC磷酸化的SN25b會負向調控囊泡釋放速率。有趣的是，在過量表現SN25b-T138A的細胞中，KCl FSK會減少其融合孔開啟的時間，顯示被PKA磷酸化的SN25b可穩定融合孔，但SN25b-S187A則否；因此，SN25b可在短時間內藉由PKA的磷酸化來調控胞吐作用的動態，此現象可能與提高SN25b與Stx1的交互作用有關。為了探討SN25b的磷酸化是否能進一步影響大規模的網路活動，我們檢測發育中大鼠視網膜內的模式化、自發性放電的現象（稱為視網膜波，此現象伴隨著週期性PKA和PKC活性的上升）。我們在突觸前神經元（星狀無軸突細胞，starburst amacrine cells，SACs）中過量表現SN25b或其突變株，利用即時鈣離子影像偵測在視網膜節神經元(retinal ganglion cells) 內鈣離子濃度瞬間的變化。在SACs過量表現SN25b，會降低視網膜波的頻率；而SACs過量表現SN25b-T138A或SN25b-S187A ，不會改變視網膜波的頻率。這些結果顯示被PKA或PKC磷酸化的SN25b可負向調控突觸前神經元神經傳導物質的釋放，進而可影響突觸後神經元的大規模網路活動。總而言之，我們的研究結果證明， SN25的單一氨基酸上的轉譯後修飾（post-translational modification），可調控神經傳導物質釋放的動態以及突觸後神經元大規模的網路活動；故細胞內訊息傳遞分子的即時變化，足以在短時間內造成神經傳導的多樣性。

關鍵字

SNAP-25 ； 磷酸化 ； 鈣離子調控的胞吐作用 ； 融合孔動態 ； 氧化電流測定術 ； kiss and run ； prespike foot ； 全融合 ； 視網膜波 ； 星狀無軸突細胞

並列摘要

The SNARE complex mediates vesicle fusion. Among three SNARE proteins, only SN25 can be phosphorylated by protein kinase A (PKA) at the residue of T138 and by protein kinase C (PKC) at the residue of S187. Previous studies showed that SN25 phosphorylation by PKA or PKC can regulate secretion via controlling the size of vesicle pool or recruiting vesicles, respectively. However, it remains unclear (1) how SN25 phosphorylation regulates the kinetics of exocytosis and (2) whether this regulation can cause a significant effect on the large-scale network activity. To address the first question, we performed single-event amperometry in PC12 cells to study the effects of SN25b phosphorylation on the exocytotic kinetics, with a special focus on the dynamics of fusion pore, reflected by foot signals preceding amperometry spikes (prespike foot, PSF). Two different secretogogues were applied to trigger Ca2+-dependent exocytosis, including KCl alone and KCl with forskolin. KCl alone reduced the secretion rate in cells overexpressing SN25b compared to control, but KCl with forskolin even more reduced the secretion rate in cells overexpressing SN25 compared to control, suggesting that SN25b may down-regulate calcium-dependent exocytosis via PKA phosphorylation. Furthermore, the secretion rate was increased in cells overexpressing the SN25b phosphodeficient mutant (SN25b-T138A or SN25b-S187A) compared to SN25b, confirming that SN25b down-regulates the secretion rate via the PKA- or PKC-phosphorylation site. Moreover, KCl with forskolin reduced PSF lifetime in cells overexpressing SN25b-T138A, but not SN25b-S187A, suggesting that SN25b PKA-phosphodeficiency destabilizes the fusion pore. Taken together, SN25b phosphorylation may regulate the kinetics of exocytosis in secretory cells. To address whether SN25b phosphorylation can cause a significant effect on the large-scale network activity, the patterned spontaneous, correlated activity (termed retinal waves) was detected in the developing rat retina where the PKA and PKC activities remain high. Live calcium imaging was subsequently performed to monitor the wave-associated calcium transients after molecular manipulation in the wave-initiating neurons (starburst amacrine cells, SACs). The frequency of retinal waves was reduced by overexpressing SN25b in SACs, whereas SN25b-T138A or SN25b-S187A in SACs did not change the wave frequency, suggesting that SN25b phosphorylation by PKA or PKC may down-regulate the wave activity from presynaptic neurons. Together, our results suggest that post-translation modification at a single residue of SN25 is sufficient to serve as a molecular regulator in modulating neurotransmitter release and the large-scale network activity.

並列關鍵字

SNAP-25 ； phosphorylation ； Ca2+-dependent exocytosis ； fusion pore kinetics ； amperometry ； kiss and run ； full fusion ； prespike foot ； retinal waves ； starburst amacrine cells

參考文獻

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