Figure 6.

The 8G5 Fab prevents inactivation of BEST1_WT. (A) The Ca²⁺-dependent inactivation of BEST1_WT and BEST1_WT–8G5 complex. Recordings were done as described in Fig. 1 A, using 50 µM Ca²⁺. For the experiment with the 8G5 Fab, after currents for BEST1_WT were obtained, 8G5 Fab was added (300 nM final concentration) to the cis and trans chambers, and the chambers were stirred for 2 min before the start of the recording. 50 μM CaCl₂ was added at the start (time = 0) of each recording. (B) Extent of inactivation in 50 µM Ca²⁺ after 10 min is plotted for BEST1_WT and the BEST1_WT–8G5 complex. Data are derived from A, and at least three separate experiments were performed to calculate SE. (C) Rescue of inactivated BEST1_WT channels by 8G5. From time = 0, currents from BEST1_WT and BEST1_S358E were recorded using 100 µM Ca²⁺ conditions. At the 5-min mark, 300 nM 8G5 Fab was added to both sides of the bilayer. At the 10-min mark, 10 mM EGTA (final concentration) was added to both sides of the bilayer to chelate free Ca²⁺. Solutions were stirred for the duration of the experiment. (D) The relative binding of 8G5 Fab to BEST1_WT and BEST1_S358E was assayed by determining the amount of free 8G5 as a function of the concentration of BEST1 in conditions containing either 10 µM Ca²⁺ (+Ca²⁺) or 5 mM EGTA (−Ca²⁺; Materials and methods). The curves correspond to fits of the following: fraction of Fab bound = [BEST1]^h/(K_d^h + [BEST1]^h), where K_d is the equilibrium dissociation constant, h is the Hill coefficient, and [BEST1] is the total concentration of BEST1. Derived K_d values are indicated.