Fig. 5. Volatile anesthetics bind the SNARE complex. ( A ) Halothane binds to the SNARE complex. Spin-spin relaxation curves for halothane (0.96 mm) in the presence of 20 μm SNARE complex or phosphate-buffered saline (PBS) buffer. Values are mean ± SD. T2s (number of independent measurements): ternary complex, 0.588 ± 0.021 s (n = 9); PBS, 0.959 ± 0.014 s (n = 8); P < 0.001 by nonlinear regression, F1,250= 121.6. ( B ) Isoflurane binds to the SNARE complex. Spin-spin relaxation curves for isoflurane (0.87 mm) in the presence of 20 μm SNARE complex or PBS buffer. T2s (number of independent measurements): ternary complex, 0.793 ± 0.0397 s (n = 3); PBS, 1.297 ± 0.0574 s (n = 5); P < 0.001 by nonlinear regression, F1,222= 55.02. ( C ) Halothane concentration versus T2 in the presence of STX:H6 SNARE complex. The slope of the line is not significantly different from 0. ( D ) Isoflurane concentration versus T2 in the presence of STX:H6 SNARE complex. The T2 at 0.23 mm not significantly different from T2 at 0.87 mm. ( E ) Lack of competition between halothane (Hal) and isoflurane (Iso) for binding to ternary complex. ( F ) Comparison of isoflurane T2s in the presence of SNARE complex containing double-His6-tagged syntaxin versus single-tagged syntaxin. *P < 0.0001 versus PBS T2. ( G ) Western blot with syntaxin antibody of fast protein liquid chromatography buffer purified SNARE complex to isolate low- and high-MW species. Peak fractions eluted from a shallow ionic strength gradient were analyzed by PAGE for SNARE complex size. Fractions containing primarily low-MW or high-MW SNARE complex were pooled, dialyzed against PBS, and separated on PAGE before staining with a syntaxin antibody. Migration of proteins of known molecular weight are shown to the left. Lanes from left to right : 1: unboiled low-MW SNARE complex pooled fractions; 2: boiled; 3: unboiled high-MW SNARE complex; 4: boiled; 5: syntaxin monomer used for SNARE complex formation. ( H ) Spin-spin relaxation curves for the low- and high-MW SNARE complexes in G . T2Low MW= 0.682 ± 0.022; T2High MW= 0.532 ± 0.021; [Isoflurane]= 0.87 mm; [Low-MW SNARE complex]= 11.9 μm; [High-MW SNARE complex]= 10.7 μm. T2Low MW> T2High MW, P < 0.001 by nonlinear regression, F1,28= 1,010; T2Low MWand T2High MW< T2PBS, P < 0.001, F1,28(Low MW vs. PBS) = 445.2, F1,28(High MW vs. PBS) = 1,566.