Each replacement of a WT subunit with a G375R mutant subunit in a BK channel acts to both left shift Vhto more negative potentials and decrease single channel conductance. (A) Single-channel currents were recorded from single channels at +100 mV after injecting the indicated cRNA. (B) Single-channel conductance g at +100 mV decreases as the number of mutant subunits increases. Mean ± SEM single-channel conductance for WT channels was 312 ± 4 pS (n = 7), decreasing to 245 ± 6 pS for hybrid channels (n = 25, P < 0.0001), and further decreasing to 190 ± 14 pS for G375R homotetrameric mutant channels (n = 8, P = 0.0003). Mean ± SEM. WT and G375R homotetrameric mutant channels were those channels expressed after injecting only WT cRNA or only G375R mutant cRNA, respectively. Hybrid channels were identified as in Fig. 4 D. (C) Plot of single channel conductance at +100 mV vs. Vh for BK channels expressed following injection of the indicated cRNA. The linear regression line plots single channel conductance g vs. Vh, where g = g(0) + aVh, where g(0) = 231.9 ± 3.9 pA, and a = 0.408 ± 0.0375 pS/mV (P < 0.0001 for slope significantly different from 0. R = 0.86). (D) Single-channel conductance for the five types of assembled channels is approximated by a linear incremental model, where each subunit contributes an increment of conductance, g(NM)=(NM)(46.1pS)+(4−NM)(76.5pS), where NM is the number of G375R mutant subunits per channel, 4-NM is the number of WT subunits per channel, g(NM) is single channel conductance as a function of NM, and 46.1 pS and 76.5 pS are the increments of g added by each mutant and WT subunit, respectively. The red dashed line indicates the predicted values of g. The homotetrameric mutant (blue) and WT (black) conductance bars are from B. The three red hybrid conductance bars plot the mean ± SEM of g for the three groups of hybrid channels identified in Fig. 5 A, where g measurements were available at +100 mV, giving: 209.0 ± 6.7 pS, n = 7; 246.0 ± 7.7 pS, n = 8; and 269 ± 2.6 pS, n = 10. For the four possible pairs of adjacent single-channel conductance bars in D, P values were calculated for g of the right conductance bar in each pair being significantly different than g for the left, and were 0.26, 0.0034, 0.0064, 0.0002, respectively; two-tailed t tests.
Figure 6.

Each replacement of a WT subunit with a G375R mutant subunit in a BK channel acts to both left shift V h to more negative potentials and decrease single channel conductance. (A) Single-channel currents were recorded from single channels at +100 mV after injecting the indicated cRNA. (B) Single-channel conductance g at +100 mV decreases as the number of mutant subunits increases. Mean ± SEM single-channel conductance for WT channels was 312 ± 4 pS (n = 7), decreasing to 245 ± 6 pS for hybrid channels (n = 25, P < 0.0001), and further decreasing to 190 ± 14 pS for G375R homotetrameric mutant channels (n = 8, P = 0.0003). Mean ± SEM. WT and G375R homotetrameric mutant channels were those channels expressed after injecting only WT cRNA or only G375R mutant cRNA, respectively. Hybrid channels were identified as in Fig. 4 D. (C) Plot of single channel conductance at +100 mV vs. Vh for BK channels expressed following injection of the indicated cRNA. The linear regression line plots single channel conductance g vs. Vh, where g = g(0) + aVh, where g(0) = 231.9 ± 3.9 pA, and a = 0.408 ± 0.0375 pS/mV (P < 0.0001 for slope significantly different from 0. R = 0.86). (D) Single-channel conductance for the five types of assembled channels is approximated by a linear incremental model, where each subunit contributes an increment of conductance, g(NM)=(NM)(46.1pS)+(4−NM)(76.5pS), where NM is the number of G375R mutant subunits per channel, 4-NM is the number of WT subunits per channel, g(NM) is single channel conductance as a function of NM, and 46.1 pS and 76.5 pS are the increments of g added by each mutant and WT subunit, respectively. The red dashed line indicates the predicted values of g. The homotetrameric mutant (blue) and WT (black) conductance bars are from B. The three red hybrid conductance bars plot the mean ± SEM of g for the three groups of hybrid channels identified in Fig. 5 A, where g measurements were available at +100 mV, giving: 209.0 ± 6.7 pS, n = 7; 246.0 ± 7.7 pS, n = 8; and 269 ± 2.6 pS, n = 10. For the four possible pairs of adjacent single-channel conductance bars in D, P values were calculated for g of the right conductance bar in each pair being significantly different than g for the left, and were 0.26, 0.0034, 0.0064, 0.0002, respectively; two-tailed t tests.

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