Using alginic acid to adsorb polypeptides at pH 2.7, we isolated a peptide pea albumin 1b (PA1b) from pea seeds. The PA1b is a single chain peptide consisting of 37 amino acid residues with 6 cysteines which constitutes the cystine-knot structure. Using microfluorometry and patch clamp techniques, we found that PA1b significantly elevated the intracellular calcium level ([Ca2+ ]i) and elicited membrane capacitance increase in the primary rat pancreatic β cells. The PA1b effect on [Ca2+]i elevation was abolished in the absence of extracellular Ca2+ or in the presence of L-type Ca2+ channel blocker, ni- modipine. Interestingly, we found that PA1b significantly depolarized membrane potential, which could lead to the opening of voltage-dependent L-type Ca2+ channels and influx of extracellular Ca2+, and then evoke robust secretion. In this study we identified the plant peptide PA1b which is capable of affecting the excitability and function of mammalian pancreatic β cell.
HU ZhiTao1, DUN XinPeng1, ZHANG Ming1, ZHU HongLiang1, XIE Li1, WU ZhengXing1, CHEN ZhengWang1 & XU Tao1,2 1 Joint Laboratory of Institute of Biophysics and Huazhong University of Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane,which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation,docking and priming along the exo-cytotic pathway play important roles in neurotransmitter release and its regulation. In the present report,we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope(TIRFM) to monitor the docking and fusion of synaptic-like microvesicles(SLMVs) in PC12 cells stimulated by high K+. Our results show that "kiss and run" is a dominative fu-sion mode in PC12 cells under high K+-challenge,and the dwell time of SLMVs is prolonged by the high K+ stimulation that suggests an enhancement of vesicle priming.
ZHOU Wei ZHU Dan LIANG Tao LI ChenHong WU ZhengXing
