Fig. 3. Volatile anesthetics bind to SNAP-25 homomers. ( A ) Spin-spin relaxation curves for halothane (0.96 mm) in the presence of 20 μm SNAP-25 or phosphate-buffered saline (PBS) buffer. T2s: SNAP-25, 0.47 ± 0.01 s (n = 1); PBS, 0.936 ± 0.012 s (n = 5); P < 0.001 by nonlinear regression, F1,90= 585.4. ( B ) Spin-spin relaxation curves for isoflurane (0.87 mm) in the presence of 20 μm SNAP-25 or PBS buffer. T2s: SNAP-25, 0.869 ± 0.071 s (n = 8); PBS, 1.30 ± 0.057 s (n = 5); P < 0.01 by nonlinear regression, F1,202= 10.98. ( C ) Western blot with rat SNAP-25 antibody of nondenaturing and denaturing PAGE of eight different SNAP-25 preparations. Aliquots from the same preparation were loaded onto each gel in the same order. ( D ) T2 values plotted against isoflurane concentration for SNAP-25 preparations containing mostly multimer (preparation 2) and mostly monomer (preparation 8). ( Inset ) The multimer curve is shown on a different scale and was fit by a four-parameter logistic equation: Kd apparent = 330 ± 120 μm; slope = 1.86 ± 1.06; Ymax = 0.365 ± 0.045 s. The curve was constrained to a Y minimum of 0.1 s. Iso = isoflurane. ( E ) Spin-spin relaxation data used to calculate the T2s in D . Note that the array sampling times for 0.13- and 0.87-mm multimers, all monomer, and PBS data are different from all other arrays, which are 0.004, 0.056, 0.1, 0.148, 0.2, 0.276, 0.348, 0.448, 0.6, 0.78, 0.9, 1.2, 1.4, 1.6, 2.0 s. The array for 0.13 mm is 0.004, 0.008, 0.016, 0.032, 0.064, 0.08, 0.1, 0.2, 0.16, 0.2, 0.256, 0.3, 0.448, 0.516, 0.6 s. The arrays for 0.87 multimer, monomer, and PBS are 0.004, 0.056, 0.1, 0.2, 0.348, 0.448, 0.6, 0.78, 0.9, 1.2, 1.4, 1.6, 2, 2.4, 3, 4 s.