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. 2008 Sep;132(3):351–360. doi: 10.1085/jgp.200809984

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© 2008 Anderson and Blaustein

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 7. — Comparison of the rate of LF_N translocation through reduced and oxidized S312C channels. (A) Reduced channels: S312C channels incorporated into the bilayer under reducing conditions (cis 250 μM TCEP) are exposed to cis LF_N (7.2 nM); this leads to block of 95% of the conductance. Free LF_N is then washed out of the cis compartment during the break in the record (250 s), and the pH is adjusted to 5.6 (cis_5.6/trans_6.6) while holding at +1 mV to prevent LF_N from going through the channel. Translocation of LF_N to the trans compartment is initiated by pulsing to +20 mV, and its rate is determined by following the rise in membrane conductance. (B) Same procedure as in A, except channels were oxidized before LF_N addition as described in Fig. 6. The increased noise is a reflection of the marked decrease in conductance after oxidation and the increased gain. The current at 1 mV seen after the break in the record (270 s) reflects a small degree of unblocking of LF_N occurring during the break. LF_N translocation through oxidized channels at +20 mV proceeds much more slowly than through reduced channels. (The instantaneous current seen in pulsing from +1 mV at +20 mV is nearly ohmic, but there is rapid reblock by LF_N that is not resolvable at this timescale.) Inset shows translocation progress on longer time scale. (C) The addition of 10 mM DTT to both sides of the bilayer restores both the conductance and gating properties of the channels. The recording is from the same bilayer as B and begins 185 s after the end of the inset in B. Break in the record is 155 s. Although not shown, the rate of LF_N translocation is also restored after DTT exposure.