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. 2013 Oct;142(4):381–404. doi: 10.1085/jgp.201311015

Figure 9.

Figure 9.

The distal section of the RCBM sequence contributes to inactivation of ANO 2 channels. (A) I/V relations recorded from 1-s voltage ramps at 5-s intervals display different speed and extent of current decline over 60 s in two cells expressing ANO 2 double mutants m15,20. (B) Four examples of current decline in ANO 2 channels with mutations in the distal segment. Inactivation kinetics exhibit large variability between individual cells. (C) The extent of inactivation, expressed as the ratio of the current amplitudes at the end (Ie) and at the start (Is) of inactivation, differs strongly between wt ANO 2 and the ANO 2 mutant m15,20; the latter exhibits impaired inactivation. Mean Ie/Is values are 0.039 ± 0.01 (n = 11) for ANO 2, 0.4 ± 0.18 (n = 6) for ANO 2 m15,20, and 0.74 ± 0.1 (n = 11) for ANO 1. **, P < 0.01; *, < 0.05; unpaired t test). Significance values were calculated for data connected by broken lines. Error bars indicate mean ± SEM. (D) Ca2+ preloading experiments (compare with Fig. 4 A) with 7.5 µM free Ca2+ at −70 mV show that inactivation of ANO 2 channels is suppressed by 20 µM TFP or 50 µM J-8, two CaM inhibitors, added to the bath solution. (E) ANO 1/ANO 2 chimaeras constructed to transfer inactivation to ANO 1. Integrating the N-terminal region of ANO 2 (red) into an ANO 1 background (gray) produced a functional but noninactivating channel, as demonstrated by the I/Vm curves recorded in 5-s intervals after the onset of the whole-cell configuration (bottom). (F) A chimera with an ANO 2 part extending from the N terminus to the position N515 of ANO 1 displayed inactivation with a time constant of 18.3 ± 3.1 s (3 cells) at −70 mV.