Biochemistry Article Figure 5. Rate of modiﬁcation by MTSES and MTSET. (A and B) Example time courses of macroscopic currents (measured at −50 mV) carried by I942C (A) and I947C (B). Current ampliutes were measured every 6 s after attainment of stable current amplitude following activation by PKA (20 nM) and ATP (1 mM). In each case, 200 μM MTSES (black bars) or MTSET (white bars) was applied to the cytoplasmic face of the patch at time zero. The decline in current amplitude due to MTS modiﬁcation has been ﬁt by a single-exponential function in each case. Currents remaining in the presence of MTSES or MTSET were inhibited by application of 5 μM CFTRinh-172 as indicated. (C) Calculated modiﬁcation rate constants for both MTSES (●) and MTSET (○) for each of the 14 MTS reagent-sensitive mutants listed. Note that T943C, V944C, and R975C were inhibited by MTSES but not by MTSET (see Figure 4). Means of data from three patches are shown. Asterisks indicate a signiﬁcant diﬀerence between the modiﬁcation rate constant for MTSES and that for MTSET (p < 0.05). macroscopic current amplitude due to MTSES modiﬁcation observed in Figure 4. ■ DISCUSSION Of 22 cysteine mutations introduced into CL3, 21 appeared to be associated with a signiﬁcant defect in protein processing relative to the Cys-less background, as determined by densitometric analysis of production of mature CFTR protein (Figure 2). Superﬁcially, this might appear to support an important, and relatively non-site-speciﬁc, role for CL3 in normal CFTR processing (see the introductory section). However, Cys-less CFTR itself has a severe protein misprocessing defect32,34,35 that in this study has been partially ameliorated by a rescue mutation in NBD1 (V510A) and by growing transfected BHK cells at a reduced temperature of 27 °C (see Materials and Methods). Thus, the relevance of these ﬁndings for the processing of wild-type CFTR is uncertain, as is the rationale for studying traﬃcking in a Cys-less CFTR background. Interestingly, many CL3 mutants could be studied function- ally in spite of an apparent lack of mature protein (Figure 2). We believe this most likely reﬂects the sensitivity of our patch clamp assay to the presence of even very small amounts of CFTR in the cell membrane. Only six mutants (S945C, L948C, H949C, K951C, K978C, and D979C) failed to generate functional channels in inside-out membrane patches (Figure 4). Each of these six was associated with undetectable Band C protein by Western blotting (Figure 2), consistent with the failure of traﬃcking of these mutants to the cell membrane. Misprocessing of CF-associated mutants S945L, H949Y, and D979A has previously been demonstrated,17,20 perhaps indicating that these residues are particularly critical for normal protein processing. However, because our work failed to show a direct correlation between protein processing determined biochemically (Figure 2) and function determined electro- physiologically (Figure 4), we cannot rule out the possibility that the six nonfunctional mutants are present in the cell membrane but nonfunctional for other reasons, such as a failure to open in response to ATP and PKA or an undetectably low single-channel conductance. Interestingly, one mutant that failed to express functional channels in our hands (K978C) has previously been studied in a Cys-less background by patch clamp recording by others18 3976 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981