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Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated …

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- SNAREs in Opposing Bilayers Interact in a Circular Array to Form Conducting Pores

Neurotransmission involves the sequential interaction of proteins in opposing bilayers (Söllner et al., 1993a,b; Rothman, 1994; Jeong et al., 1998). The classical concept of fusion is a three-step process of cell excitation, docking, and fusion, in which docking may occur before cell excitation. Fusion has been implicated to occur via soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) (Weber et al., 1998). SNAREs are classified as v-SNARE and t-SNAREs, depending on their primary location either in vesicles (v-) or in target (t-) membranes (Rothman, 1994). Studies demonstrate that t- and v-SNAREs reconstituted into lipid vesicle membranes can fuse with one another, suggesting SNAREs to be the minimal membrane fusion machinery (Weber et al., 1998). The structure of the SNARE complex formed by interacting native (Jeong et al. 1998) and recombinant (Hanson et al., 1997; Sutton et al., 1998) t- and v-SNAREs, has been examined using electron microscopy (Hanson et al., 1997; Jeong et al., 1998) and x-ray crystallography (Sutton et al., 1998). However, the morphology and arrangement of SNAREs in lipid bilayers and their interaction and arrangement when associated within opposing bilayers, remains to be established.

Here we examine the structure and arrangement of purified recombinant t- and v-SNAREs in artificial lipid bilayers, using atomic force microscopy (AFM). To further evaluate the functional properties of SNARE proteins in bilayers, conductance and capacitance of membranes in the presence and absence of SNARE proteins was examined (Cohen and Niles, 1993; Kelly and Woodbury, 1996; Woodbury, 1999). If pore structures were to form by direct addition of SNAREs to a single membrane, an increase in conductance would be observed due to the movement of ions through the pore. To determine the interaction between t- and v-SNAREs present in opposing bilayers, we used v-SNARE reconstituted artificial lipid vesicles and challenged them with t-SNARE reconstituted lipid membranes. The structure and arrangement of the SNARE complex formed as a result, and any changes in capacitance or conductance were recorded using AFM (Schneider et al., 1997; Cho et al., 2002a,b,c) and a bilayer apparatus (Woodbury and Miller, 1990).

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