1. -140 mV to -80 mV -140 mV to -60 mV -140 mV to -50 mV -140 mV to -100 mV (n=12 ) (n=9) (n=20 ) (n=26 ) (n=12 ) # of channels = 8

2. -140 mV to -40 mV -140 mV to +30 mV -140 mV to +40 mV -140 mV to +60 mV (n=18 ) (n=12 ) (n=13 )

3. -140 mV to +80 mV -140 mV to +90 mV -140 mV to +100 mV -140 mV to +110 mV (n=23 ) (n=21 ) (n=15 ) (n=14 )

4. -140 mV -100 mV -80 mV +40 mV + 80 mV + 100 mV -40 mV -140 mV

5. Previously reported as 89pS (20140827), we find out it is closer to be 60.7 pS (20150423). This discrepancy can be somewhat explained by the program algorithm in Clampfit which requires two consecutive flat lines. With high amount of NaChBac channels, this is difficult to observe, and when it is observed, it is generally when two channels are closed at the same time. what the program looks forwhat tends to be the case Studer (2009) reports 120 pS with 150 mM NaCl, 20mM NaCH3CO2. His bilayer was POPE:POPC (3:7). pA ms pA ms

6. 150 mM NaCl in Cis and Trans Filter: 8 pole Bessel 1000 Hz cutoff Sampling interval: 50µs -20pA/-2.5pA = 8 protein channels

7. Cis and trans chamber are filled with either 1) 150 mM NaCl, 10mM Tris 7.4 2) 150 mM KCl, 20 mM HEPES 7.4 Recorded 1 second after voltage was set P Na /P K = 171 ± 16 (n = 8) from Ren et al., 2001

8. 150 mM KCl in Cis and Trans ~ 60.8pA/5.9pA = 10.3 channels

9. 150 mM KCl Cis/Trans Cis / trans blocker: Ni+ recorded 1 second after voltage 150 mM KCl Cis/Trans +60 and -60 mV voltage applied over 10 seconds