The Sec61p Complex Is a Dynamic Precursor Activated Channel Andreas Wirth, Martin Jung, Christiane Bies, Michael Frien, Jens Tyedmers, Richard Zimmermann, Richard Wagner  Molecular Cell  Volume 12, Issue 1, Pages 261-268 (July 2003) DOI: 10.1016/S1097-2765(03)00283-1

Figure 1 The Sec61p Channel in RM Vesicles and Sec61p Proteoliposomes (A–F) RMs + puromycin. (A) Current recordings from a bilayer containing four active channel units at a holding potential of Vm = 35 mV in symmetrical buffer containing 10 mM Mops/Tris (pH 7.0) and 250 mM KCl, (symmetrical standard buffer). Lower trace scale expanded view of the shaded part from the upper current recording. Arrows indicate direct transitions between different current levels. (B) Mean variance plot of the current recording from part (A) depicting direct gating transitions between different open channel amplitudes. (C) Amplitude histogram of the current recording in part (A). (D) Current recordings from a bilayer containing nine active channel units at a holding potential of Vm = –15 mV in symmetrical standard buffer. Lower trace scale expanded view of the shaded part from the upper current recording. (E) Mean variance plot of the current recording from part (D) depicting direct gating transitions between different open channel amplitudes. (F) Amplitude histogram of the current recording in part (D). (G) SDS page of the purified Secc61p complex. (H–J) Proteoliposomes containing the purified Sec61p complex + ppcecA. (H) Current recordings from a bilayer containing a single active channel unit at a holding potential of Vm = –85 mV in symmetrical standard buffer. Lower traces scale expanded views of the shaded part from the upper current recording. (I) Mean variance plot of the current recording from part (H) depicting direct gating transitions between different open channel amplitudes. (J) Amplitude histogram of the current recording in part (H). Molecular Cell 2003 12, 261-268DOI: (10.1016/S1097-2765(03)00283-1)

Figure 2 Distribution of Open Channel Amplitudes Observed for the Sec61p Channel (A–C) Current recordings were performed under symmetrical conditions using the standard buffer. Data were binned with a width of 10 pA. The dashed line shows the Gauss-fit of the data. (A) RMs + puromycin. Data were collected from n = 15 independent bilayers. Values were normalized with the total number of gating events being n = 10533. (B) PKRMs + ppcecA. Data were collected from n = 5 independent bilayers. Values were normalized with the total number of gating events being n = 418. (C) Proteoliposomes containing the purified Sec61p complex + ppcecA. Data were collected from n = 5 independent bilayers. Values were normalized with the total number of gating events being n = 566. (D) Probability of the Sec61p channel being in any of its open states at given membrane voltages. Current recordings were performed under symmetrical conditions using the standard buffer. Data were calculated from bilayers containing a single active copy of the Sec61p channel. At a given membrane voltage Popen was calculated from the quotient of the measured mean current during a 60s recording and the maximal current as calculated from the chord conductance of the mean Popen = I60smeanV/Ichord conduct.maxV. Molecular Cell 2003 12, 261-268DOI: (10.1016/S1097-2765(03)00283-1)

Figure 3 Exotoxin A Blocks Channel Openings with Large Amplitudes (A) Time course of the ExotoxinA experiment. Current recordings of a single active Sec61p channel (Vh = 25 mV). Current recordings were performed under symmetrical conditions using the standard buffer. The course of the applied voltage addition of Exotoxin A and stirring of the cis compartment (cytosolic side) are indicated. (B) Amplitude histogram of the current recording in (A) before addition of Exotoxin A. (C) Amplitude histogram of the current recording in (A) after addition of Exotoxin A. (D and E) Frequency of the different channel amplitudes observed for the Sec61p channel in the absence (D) and presence (E) of 30 nM Exotoxin A on both sides of the membrane. Current recordings were performed under symmetrical conditions using the standard buffer. Data binned with a width of 10 pA were collected from n = 3 independent bilayers. Values were normalized with the total number of gating events being n = 220. The dashed line shows the Gauss-fit of the data. Molecular Cell 2003 12, 261-268DOI: (10.1016/S1097-2765(03)00283-1)

Molecular Cell 2003 12, 261-268DOI: (10.1016/S1097-2765(03)00283-1)