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Original Article

Covalent Modification of Cysteine-Substituted Channels Expressed in Xenopus Oocytes Shows That Activation Is Due to the Opening of Channels Resident in the Plasma Membrane

Xuehong Liu^a,b, Stephen S. Smith^a,b, Fang Sun^b, and David C. Dawson^a,b

^a Oregon Health Sciences University, Portland, OR 97201
^b Department of Physiology, University of Michigan, Ann Arbor, MI 48109
Department of Physiology/Pharmacology, L334, 3181 SW. Sam Jackson Park Road, Portland, OR 97201.(503) 494-4352

dawsonda{at}ohsu.edu

Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P_o), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET⁺). Covalent modification of R334C CFTR with MTSET⁺ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET⁺ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET⁺. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of 24–48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.

Key Words: trafficking • MTS reagents • labeling • mutagenesis • oocyte expression

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