Figure 7.
Figure 7. (A and B) Testing the state dependence of appearance of a constitutive component in oocytes expressing Q1(-Cys)-I145C/E1-G40C after DTT washout. Data from two experiments are shown, with time courses of appearance of the constitutive component (Istep) shown on the left, and superimposed current traces shown on the right. In both experiments, before DTT treatment, the oocytes manifest slowly activating currents without any constitutive component (dotted traces in right panels). The oocytes are exposed to DTT 10 mM for 10 min (gray shade, time axis breaks between start of DTT application and last few data points in DTT). In the presence of DTT, the current amplitudes increase but the currents remain slowly activating without a significant constitutive component (gray traces in the right panels). In A, during DTT washout the oocyte is repetitively pulsed from Vh −100 to +60 mV for 2 s, followed by a step to −60 mV for 2 s, once every minute (voltage clamp diagram in inset). The appearance of Istep follows a single exponential time course, with a time constant of 3.1 min (black thick curve superimposed on the open circle data points). In B, during DTT washout the oocyte is held at Vh −100 mV without pulsing for 15 min. The current induced by the first pulse after resuming pulsing is highlighted by the thick black current trace (right) and the small Istep component during the first pulse is highlighted by a solid black circle (left). The dotted curve is a scaled single exponential time course similar to that shown in Fig. 7 A. (C) Testing whether membrane depolarization by elevating [K]o facilitates disulfide bond formation between Q1 145C and E1 40C. COS-7 cells are transfected with cDNA(s) listed on the top two rows. Cells are incubated in medium containing 5 mM [K]o for 48 h. Cells in lanes 4 and 5 are treated with 10 mM DTT for 10 min to break disulfide bonds, followed by thorough rinsing to remove DTT. Cells in lane 4 are incubated in 5 mM [K]o medium, while cells in lane 5 are incubated in 100 mM [K]o medium for 10 min. All cell preparations are treated with NEM to protect free thiol groups, followed by whole cell lysis. Whole cell lysates are run on nonreducing SDS gel and probed for Q1 with V5 mAb.

(A and B) Testing the state dependence of appearance of a constitutive component in oocytes expressing Q1(-Cys)-I145C/E1-G40C after DTT washout. Data from two experiments are shown, with time courses of appearance of the constitutive component (Istep) shown on the left, and superimposed current traces shown on the right. In both experiments, before DTT treatment, the oocytes manifest slowly activating currents without any constitutive component (dotted traces in right panels). The oocytes are exposed to DTT 10 mM for 10 min (gray shade, time axis breaks between start of DTT application and last few data points in DTT). In the presence of DTT, the current amplitudes increase but the currents remain slowly activating without a significant constitutive component (gray traces in the right panels). In A, during DTT washout the oocyte is repetitively pulsed from Vh −100 to +60 mV for 2 s, followed by a step to −60 mV for 2 s, once every minute (voltage clamp diagram in inset). The appearance of Istep follows a single exponential time course, with a time constant of 3.1 min (black thick curve superimposed on the open circle data points). In B, during DTT washout the oocyte is held at Vh −100 mV without pulsing for 15 min. The current induced by the first pulse after resuming pulsing is highlighted by the thick black current trace (right) and the small Istep component during the first pulse is highlighted by a solid black circle (left). The dotted curve is a scaled single exponential time course similar to that shown in Fig. 7 A. (C) Testing whether membrane depolarization by elevating [K]o facilitates disulfide bond formation between Q1 145C and E1 40C. COS-7 cells are transfected with cDNA(s) listed on the top two rows. Cells are incubated in medium containing 5 mM [K]o for 48 h. Cells in lanes 4 and 5 are treated with 10 mM DTT for 10 min to break disulfide bonds, followed by thorough rinsing to remove DTT. Cells in lane 4 are incubated in 5 mM [K]o medium, while cells in lane 5 are incubated in 100 mM [K]o medium for 10 min. All cell preparations are treated with NEM to protect free thiol groups, followed by whole cell lysis. Whole cell lysates are run on nonreducing SDS gel and probed for Q1 with V5 mAb.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal