Effect of Simple Anesthetics on SNARE Fusion Proteins and on Fusing Membranes

Abstract

For over 170 years, simple molecules like ether have been used to induce anesthesia in humans. Nevertheless, the mechanisms of action for many general anesthetics are not known. Here we explore the effect of short chain alcohols and diethyl ether on the folding of SNARE proteins, known to drive exocytosis in neurons, and also on membrane-membrane fusion, the final step in neurotransmitter release. The SNARE proteins SNAP-25, syntaxin, and VAMP provide the four helical regions (SNARE domains) that form a coiled-coil complex required for neurotransmitter release. This complex is continually formed and unwound as exocytotic vesicles fuse and are recycled. Circular Dichroism was used to measure secondary structure of SNAP-25 and its two SNARE domains following addition of alcohols and ether. We observed an increase in α-helical structure when methanol is added or the temperature is lowered. This increase in helicity occurred for each domain and for the entire protein. This is similar to the helical shift observed when SNAP-25 forms a complex with syntaxin and VAMP. We utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a membrane in the absence of SNARE proteins. Changes in fusion rates were observed in response to increasing doses of ether or short-chain alcohols on one or both sides of the membrane. Alcohols applied to the trans side of the membrane (side opposite the vesicles) inhibited fusion at physiologically-relevant doses (Paxman et al. 2017. Biophys J. 112:121-132.). Therapeutic doses of ether also decreased fusion rates when added trans. These data show that some anesthetics may induce their effects by altering the SNARE fusion machine and/or raising the activation energy of membrane-membrane fusion, decreasing neurotransmitter release.