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. 2006 Oct 12;25(20):4728–4739. doi: 10.1038/sj.emboj.7601373

Figure 2.

Figure 2

Expression and function of cysteine-deficient CFTR channels in Xenopus oocytes. (A) Two-microelectrode voltage-clamp current recordings from uninjected oocyte and oocytes expressing WT CFTR (2.5 ng cRNA) or HA-tagged Cys-free CFTR 16CS+C590L/C592L (20 ng cRNA); vertical current deflections monitor conductance, which was transiently increased by brief exposure to 40 μM forskolin (between arrows). (B) Summary of mean±s.d. whole-oocyte conductances determined as in (A), before (‘resting', white bars) and at maximal forskolin effect (‘stimulated', gray bars), 3 days after injection of 20 ng cRNA encoding HA-tagged CFTR 16CS constructs containing native C590 and C592 or substitutions at those positions as indicated; forskolin elicited significant conductance only with C590 and C592 unchanged (131±6 μS, n=3), or replaced by valines (119±6 μS, n=15) or leucines (155±9 μS, n=3). (C) Conductances from oocytes injected with 2.5 ng cRNA, and measured 1 day later for WT (153±17 μS, n=3), or 3 days later for HA-tagged 16CS mutants with C590/C592 (42±10 μS, n=6) or C590V/C592 V (51±3 μS, n=6). This under-represents the functional difference between WT and mutants, as conductance is enhanced by injecting more cRNA or allowing more time for its expression. (D) WT CFTR and Cys-free CFTR (16CS+C590V/C592V) were immunoprecipitated from membranes of oocytes injected with cRNA amounts indicated, and subjected to SDS–PAGE and Western blot analysis; arrows mark core-glycosylated and mature fully glycosylated CFTR.