Characterization of a noninactivating mutant, T220A. (A) A side view of the NaChBac homology model highlighting the location of T220 of the S6 segment (left). The structure is also shown with a top view (right) that is sliced approximately through the middle of the pore from the side parallel to the membrane, then rotated 90° horizontally to highlight the orientation of T220. (B) Mutation of T220A of NaChBac has resulted in a noninactivating channel. Normalized traces of wild-type NaChBac and T220A mutant are shown. Cells were held at a holding potential of −120 mV, and channels were activated by 500 ms of depolarization pulse to −20 mV. The dashed line indicates the zero current level. Bar, 250 ms. (C) Representative current traces of T220A mutant. HEK-293 cells were held at a holding potential of −120 mV with patch-clamp configuration. NaChBac channels expressing T220A were activated by 1 s of depolarizing pulses from −100 to 0 mV in 10-mV increments. Bar: horizontal, 250 ms; vertical, 4 nA. (D) Current-voltage relationship between wild-type NaChBac and T220A mutant. A significant 20-mV left shift was observed with T220A (V_1/2 = −65.765 ± 2.89 mV; mean ± SEM, n = 5) as compared with wild type (V_1/2 = −45.52 ± 0.90 mV; mean ± SEM, n = 3), with P < 0.01. Error bars represent SEM.