Liu X , Smith SS , Sun F , Dawson DC .

DK45880 NIDDK NIH HHS , R01 DK045880 NIDDK NIH HHS , R56 DK045880 NIDDK NIH HHS

	Figure 1. I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.
	Figure 2. Modification of R334C CFTR by MTSET+ was stable for at least 5 h. The conductance (gCl@Erev) was first obtained 30–40 min after the initial exposure of the oocytes to stimulatory cocktail when the activation of the Cl− conductance had attained a steady state (control). Each oocyte was exposed to 100 μM or 1 mM MTSET+ for 20 s. The MTSET+-containing stimulatory cocktail was then replaced with stimulatory cocktail lacking the thiol reagent, and the oocytes were continuously perfused for up to 5 h. The conductances measured at 2 min, 2 h, and 5 h after exposure of oocytes to MTSET+ were normalized to the steady-state conductance before exposure to MTSET+.
	Figure 3. The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with MTSET+ before the activation. Records of gCl versus time obtained from R334C CFTR expressing oocytes that were obtained from the same frog and assayed on the same day. Oocytes were always perfused with frog Ringer's unless noted (see materials and methods). After a control period, they were perfused with stimulatory cocktail containing 10 μM isoproterenol and 1 mM IBMX (Isop + IBMX). (A) A record of gCl versus time showing that at the steady-state activation, MTSET+ caused about a doubling of gCl. (B) A record of gCl versus time showing that the activated gCl at the steady state of an oocyte preexposed to MTSET+ was much higher than the gCl at unmodified condition as seen in A, and 2-ME reduced gCl to ∼50% of the maximum gCl. (C) A record of gCl versus time of showing that the exposure to MTSES− after prelabeling with MTSET+ had no effect on gCl before and after activation.
	Figure 4. MTSET+ labeling did not affect the activation and inactivation process of R334C CFTR. A record of the conductance measured at the reversal potential throughout an experiment. After activation, an oocyte was exposed to 100 μM MTSET+, and then was inactivated without removing MTSET+. The oocyte was then reactivated, showing a gCl similar to the gCl before inactivation. The gCl was then reduced to ∼50% by 2-ME.
	Figure 5. The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with 20-s exposure to MTSET+ before the activation. (A) A record of the conductance measured at the reversal potential throughout an experiment. (B) Exposure to 1 mM MTSET+ or 1 mM MTSES− had no significant effects on the background conductance before activation of CFTR (1 and 2). (C) Modification by 1 mM MTSET+ for 20 s before activation prevented any further modification by MTSES+ or MTSET+ during or after activation (3–5), and 2-ME reversed the effect of MTSET+ (6). (D) Further modification by MTSET+ was possible after 2-ME treatment and was also reversed by 2-ME (7 and 8). The apparent lower values of Erev (∼17 mV) resulted from a shift in the tip potential of the recording electrode.
	Figure 6. Time-dependent increase in the conductance of R334C CFTR in the first 5 d after cRNA injection. Oocyte conductance in both inactive and cAMP-activated states was measured on days 1, 2, 3, 4, and 5 (24, 48, 72, 96, and 120 h) after cRNA injection and the values were normalized to the conductance on day 2.
	Figure 7. The addition of new channels can be monitored by labeling preexisting channels with MTSET+. (A) A record of the conductance measured at the reversal potential throughout an experiment on day 1. The conductance was increased after 4.5 h due to the addition of new, unlabeled channels. (B) A record of the conductance measured at the reversal potential throughout an experiment on day 5. No increase in the conductance was observed during the 4.5-h period.
	Figure 8. BFA prevented the addition of new channels on day 1, and had no effect on the conductance on day 5. Records of the conductance measured at the reversal potential throughout experiments. Oocytes were exposed to BFA throughout the entire experimental period. No increase in the conductance with time was observed after 4.5 h on either day 1 (A) or day 5 (B).
	Figure 9. Long-term BFA treatment prevented the addition of new channels. Some oocytes were kept in MBSH solution as usual (control). Some oocytes were transferred to the MBSH solution containing 5 μM BFA a few hours after cRNA injection and remained in the same solution thereafter (BFA-treated). The background conductance (gbkg) and cAMP-elevated Cl− conductance (gCl) were assayed at 24 and 48 h after cRNA injection.

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