The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an alpha-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.

The synaptic SNARE complex is a highly stable four-helix bundle that links the vesicle and plasma membranes and plays an essential role in the Ca2+-triggered release of neurotransmitters and hormones. An understanding has yet to be achieved of how this complex assembles and undergoes structural transitions during exocytosis. To investigate this question, we have mutated residues within the hydrophobic core of the SNARE complex along the entire length of all four chains and examined the consequences using amperometry to measure fusion pore opening and dilation. Mutations throughout the SNARE complex reduced two distinct rate processes before fusion pore opening to different degrees. These results suggest that two distinct, fully assembled conformations of the SNARE complex drive transitions leading to open fusion pores. In contrast, a smaller number of mutations that were scattered through the SNARE complex but were somewhat concentrated in the membrane-distal half stabilized open fusion pores. These results suggest that a structural transition within a partially disassembled complex drives the dilation of open fusion pores. The dependence of these three rate processes on position within the SNARE complex does not support vectorial SNARE complex zipping during exocytosis.

Recent experiments have shown that flux through the fusion pore is sensitive to manipulations of the side-chain size of certain residues in the syntaxin (syx) membrane anchor. These residues were proposed to line the wall of the fusion pore of Ca(2+)-triggered exocytosis. Here we continued this line of experimentation to examine possible electrostatic interactions between the pore lining residues and the neurotransmitter norepinephrine (NE). Replacing syx pore-lining residues with aspartate enhanced NE flux above that expected for the size of the aspartate side chain. In contrast, substitution with arginine reduced NE flux below that expected for the size of its side chain. Substituting aspartate and arginine into the nonpore-lining residues did not alter the fusion pore flux. Other amino acids with ionizable side chains had variable effects. These results indicate an electrostatic interaction between the pore-lining residues of syx and NE, and provide additional evidence that the syx membrane anchor is a structural component of the fusion pore.

Different organophosphorus pesticides (OPs) normally vary in the level of toxicity to human health, but common electrochemical identification methods usually require complex electrode fabrication processes and high detection cost. To overcome these problems, a pesticide residues identification method based on multilayer paper-based microfluidic chip was proposed. The microfluidic chip integrates the enzyme inhibition and a label-free screen-printed carbon electrode (SPCE). Three different pesticides were selected as study objects, respectively, avermectin, phoxim, dimethoate. This chip realizes on-chip identification via analyzing impedance time-sequence spectrum data aroused by the enzyme inhibition during the 15 min period. A classification model based on principal component analysis (PCA) and support vector machines (SVMs) was established to evaluate these data. Furthermore, the real-world sample (lettuce) was tested to verify the feasibility of the proposed detection method. The results showed that the classification accuracy was achieved around 93% in multiple experiments. Therefore, the method has good stability and specificity in rapid identification of different pesticides. Practical Applications Identification of pesticide types is essential to human health, for this, impedance time-sequence spectrum data aroused by the enzyme inhibition during the 15 min period was used to establish a classification model to identity different types of pesticides. In this article, multilayer paper-based microfluidic chip and a label-free screenprinted carbon electrode (SPCE) was proposed without complicated operation steps and expensive cost, which provide a basis for establishment of the rapid pesticide discriminator.

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