Mutation in the zinc-binding motif in M7kd disrupts the interaction between M7cd and M7kd, which attenuates Mg²⁺ inhibition of the M7cd current. (A) Expression of M7cd and M7kd in M7cd-expressing HEK293 cells transfected with M7kd-wt (lane 2), M7kd-C1809S (lane 3), M7kd-C1813S (lane 4), H1751A (lane 5), and H1808A (lane 6). M7cd expression (172 kD; arrow) is induced by doxycycline treatment. M7kd expression (40 kD; arrowhead) is induced by baculovirus infection. (B and C) The effect of M7kd-C1809S (B) or -C1813S (C) expression on M7cd current induced by baculovirus infection in the absence (open circles) or presence of 0.2 mM [Mg²⁺]_i (closed circles). Each symbol represents the mean ± SEM of 23 and 9 recordings for M7kd-C1809S and 20 and 9 recordings for M7kd-C1813S in the absence or presence of 0.2 mM [Mg²⁺]_i, respectively. (D) [Mg²⁺]_i-dependent curves of M7cd current with coexpression of M7kd-C1809S (orange squares) or -C1813S (green circles) in control conditions. The current was inhibited by intracellular free Mg²⁺ with an IC₅₀ of 8.9 µM and 8.0 µM in M7cd-expressing cells with coexpression of M7kd-C1809S and -C1813S, respectively. Each symbol represents the mean ± SEM of 23, 28, 47, 5, and 9 recordings for M7kd-C1809S and 21, 16, 24, 5, and 9 recordings for M7kd-C1813S in the absence or presence of 0.8 µM, 2.8 µM, 20.9 µM, and 0.2 mM [Mg²⁺]_i, respectively. The dashed lines are the [Mg²⁺]_i-dependent curve of the M7cd current with (black) or without (red) coexpression of M7kd-wt (from Figs. 4 C and 5 D). (E) The time course of the current in M7kd-C1809S–coexpressing cells in the presence of 2.8 µM [Mg²⁺]_i with (red circles) or without (black squares) H₂O₂ application at 2 min. Each symbol represents the mean ± SEM (vertical bar) of 20 recordings. Similar to cells expressing M7cd alone (Fig. 4 A, blue squares), the current decreased over time even under control conditions (black squares), and repeated-measures ANOVA revealed that H₂O₂ had no significant effect on the current decrease (degrees of freedom were corrected using the Greenhouse-Geisser estimate of epsilon; P = 0.164). (F) M7kd-C1809S expression did not affect the M7cd-S1107E current. Each symbol represents the mean ± SEM of six recordings. (G and H) The effect of M7kd-H1750A (G) or -H1807A (H) expression on M7cd current by baculovirus infection in the absence (open circles) or presence of 0.2 mM [Mg²⁺]_i (closed circles). Each symbol represents the mean ± SEM of 9 and 8 recordings for M7kd-H1750A and 9 recordings for M7kd-H1807A in the absence or presence of 0.2 mM [Mg²⁺]_i, respectively. (I) Summary of M7cd current densities at 2 min after break-in in M7kd-wt–, -C1809S–, -C1813S–, -H1750A–, or -H1807A–coexpressing cells. Each bar represents the mean ± SEM of 21, 8, 9, 9, 8, and 9 recordings in cells expressing M7cd alone and M7cd with M7kd-wt, -C1809S, -C1813S, -H1750A, and -H1807A, respectively.