Correlation of UDCA membrane binding and rBASIC activation.
A) Confocal images of patch pipettes containing outside-out patches excised from Xenopus laevis oocytes. Left panel, membrane patch excised from an oocyte expressing rBASIC, before (upper panel) and after (lower panel) application of UDCA-NBD/UDCA (50 µM/1.6 mM). Right panel, membrane patch excised from an uninjected control oocyte, before (upper panel) and after (lower panel) application of UDCA-NBD/UDCA. Green fluorescence signals originated from UDCA-NBD binding to the membrane, red fluorescence signal originated from DY647 background dye (1 µM) staining the bath solution. The transmission channel is overlayed. Scale bar = 5 µm. B) Binding time courses and current time courses after sudden UDCA-NBD/UDCA concentration jumps, obtained from a membrane patch excised from an rBASIC expressing oocyte (left panel) and a membrane patch excised from an uninjected control oocyte (right panel). Green traces represent fluorescence signals induced by UDCA-NBD binding and unbinding to the patch membranes (for clarity, increase in fluorescence is depicted as downward deflection). Grey traces represent simultaneously recorded current traces. Averages of three consecutive measurements are shown. Time courses of activation/binding and deactivation/unbinding were fitted by the sum of two exponentials (equations (2a) and (2b)). τfast, τslow represent fast and slow time constants for rBASIC activation/inactivation and UDCA-NBD binding/unbinding events (a, rBASIC activation; b, UDCA-NBD binding; d, rBASIC deactivation; u, UDCA-NBD unbinding). C) and D) Quantitative comparison of slow and fast time constants of rBASIC activation and UDCA-NBD binding (C) and rBASIC deactivation and UDCA-NBD unbinding (D). Error bars represent SEM; n = 3–7.