Effect of mambalgin-2 on the pH-dependent activation and inactivation of different chimera currents.
A, inactivation ratios (I/Imax) for ASIC1a were calculated in the absence or presence of toxin after stimulation at pH 5.0 from holding pH 8.0 (Imax) and holding pH 7.1 (I), a value chosen in the descending part of the curves (a or a′). Activation ratios (I/Imax) for ASIC1a were calculated in the absence or presence of toxin from holding pH 8.0 and after stimulation at pH 4.0 (Imax) and pH 6.2 (I), a value selected in the ascending part of the curves (b or b′). Protocols used to generate the inactivation (inacti) and activation (acti) curves are shown in the inset, i.e. variable holding pH values and test pH 5.0 for inactivation and holding pH 8.0 and variable test pH values for activation. B–E, inactivation and activation ratios for ASIC1a (B) and different chimeras (C–E). Test and holding pH values for the different chimeras were chosen to be in the ascending or descending portion of the activation and inactivation curves, respectively, based on their properties shown in A (for ASIC1a) and in Table 1 and Fig. 2. Protocols are indicated in the inset with short horizontal lines indicating variable holding pH and unique test pH (for inactivation) and variable test pH from a unique holding pH (for activation). Recordings with or without mambalgin-2 (400 nm; except for ASIC1a 200 nm) were made on the same oocyte (paired measures). Error bars represent S.E. F, ratios corresponding to data shown in B–E. The pH-dependent inactivation is not significantly affected by mambalgin-2 in the different chimeras, but the pH-dependent activation is shifted toward more alkaline pH (chimeras 1a/2aP+RDQ-KQE and 2a/1aT) or more acidic pH (chimera 2a/1aT+P). This shift of the activation curve toward more acidic or more alkaline pH represents stabilization or destabilization of the closed state of the channel, respectively, and explains in large part inhibition or potentiation by the toxin. ns, no significant shift of the curve; Tx, toxin mambalgin-2.