Fig. 6.
Evidence that the N terminus of LFN enters the channel formed by (PA63)7.(A and B) LFN (0.6 μg) containing a biotin group disulfide-linked either to the N (A) or the C(B) terminus was incubated with 6 μg of streptavidin at room temperature for 10 min before being added to the cis compartment. The membrane-impermeant disulfide reducing agent Tris-carboxyethylphosphine (TCEP) (5 mM) was added as indicated. The voltage was held at +20 mV throughout the experiment. (C) Trans addition of Ni2+ slowed unblocking of N-terminal H6-tagged LFN. EDTA was omitted from the buffer. We added 500 μMNi2+ and 1 mM EDTA to the trans compartment at indicated time points. Although some of the channels became immediately unblocked at –20 mV, others took longer to unblock. This delay may be a consequence of the distribution of LFN penetration of channels at a given voltage. (D) Use of biotin-LFN-DTA to demonstrate penetration of the N terminus to the trans compartment. The buffer used was the same as in Fig. 3, except that 1 mM adenine was present. Eleven minutes before starting the recording, 2 ng of (PA63)7 was added to the cis compartment held at +20 mV. Then, biotin-LFN-DTA (4 nM) was added to the same compartment. At the break, the cis compartment was perfused with 10 ml of buffer, and 10 μg of streptavidin was added to the trans compartment, as indicated. The voltage was shifted to –20 mV, and TCEP (5 mM) was added to the trans compartment, as indicated. The small response at –20 mV before TCEP addition may reflect a small amount of protein not binding streptavidin. The traces shown in A, B, and C were representative of three, four, and four independent trials, respectively, all of which were essentially identical. The trace in D is representative of six trials, all but one of which were successful.