FIGURE 3.
Single channel characteristics of ATOMwg produced by cell-free expression in a wheat germ system. A, ATOMwg was purified via its C-terminal His6 tag using Ni-NTA. Total translation reaction (T), unbound fraction (U), and 2-fold concentrated eluate (E) were analyzed by SDS-PAGE (top) and immunoblot using α-His-HRP (bottom). The migration position of ATOMwg is indicated by an arrowhead. The impurity (double band ∼50 kDa) marked with an asterisk is a constituent of the wheat germ extract known to be co-purified by Ni-NTA. B, single channel current traces reveal complete channel closure in three distinct steps marked by horizontal dashed lines, with O3, O2, and O1 for the number of open pores and C for the closed state of the complete trimeric channel unit. C, the voltage-dependent gating behavior is reflected in the frequency distributions of gating transition amplitudes (n = 3 single channels; τ > 0.1 ms): 110 ± 20, 180 ± 15, 380 ± 100, 475 ± 25, and 570 ± 20 pS at positive voltages and 130 ± 35, 245 ± 30, and 330 ± 20 pS at negative voltages. Buffer conditions were symmetric with 250 mm KCl, 10 mm MOPS/Tris, pH 7, in cis and trans.