Biochemistry Figure 2. Protein expression of CL3 mutants. (A) Representative Western blot for CFTR using protein from BHK cells transfected with the wild type, the Cys-less mutant, or the named cysteine mutants in a Cys-less background. Note the near absence of Band C protein for these mutants. (B) Mean abundance of Band C protein (as a percentage of total). Only R975C was not signiﬁcantly diﬀerent from the Cys-less mutant (p > 0.6); for H950C and L983C, p < 0.05, and for all other mutants, p < 0.00005. Means of data from nine independent transfections for Cys-less and three to six for other constructs are shown. the presence of the V510A “rescue” mutation.32 In contrast to the Cys-less background, almost all CL3 cysteine mutants studied gave almost undetectable levels of Band C protein (Figure 2), suggesting that mutations in this juxtamembrane region result in serious misprocessing of the CFTR protein. Eﬀects of the MTS Reagents on Macroscopic Currents. Functional properties of mutant CFTR expressed in BHK cells were investigated using inside-out patch recording. Sixteen of the 22 mutants that were constructed generated macroscopic PKA- and ATP-dependent currents in inside-out membrane patches (e.g., Figure 3). However, for the six remaining mutants (S945C, L948C, H949C, K951C, K978C, and D979C), application of PKA and ATP consistently failed to generate a signiﬁcant increase in the macroscopic current amplitude above background levels. Each of these six mutants was also associated with undetectable levels of mature, Band C protein (Figure 2), suggesting that these mutants were misprocessed and absent from the cell membrane. However, other CL3 mutants with similar apparent misprocessing as determined by Western blotting (Figure 2) did generate measurable macroscopic currents. Our favored interpretation of this apparent discrepancy is that our patch clamp experiments are, in fact, able to detect tiny amounts of CFTR protein in the cell membrane that cannot be detected by Western blotting, either because the amount of mature Band C protein is below detection levels or because some immature Band B protein is present at the membrane. However, on the basis of our results, Article we cannot rule out the alternative explanation that S945C, L948C, H949C, K951C, K978C, and D979C are also present at the cell membrane but that these mutants generate CFTR channels that are functionally nonresponsive to PKA and ATP. For those 16 CL3 mutants that could be studied functionally, modiﬁcation of introduced cysteines by negatively charged MTSES and positively charged MTSET was investigated by application of these reagents to the cytoplasmic face of inside- out membrane patches following channel activation by PKA and ATP. Three diﬀerent types of functional eﬀects were observed. In most cases (L941C, I942C, K946C, I947C, H950C, L973C, N974C, S977C, I980C, A981C, and I982C), macroscopic currents were inhibited by both MTSES and MTSET (e.g., Figure 3A). In contrast, T943C, V944C, and R975C were inhibited by MTSES but not signiﬁcantly aﬀected by MTSET exposure (e.g., Figure 3B). Two mutants, F976C and L983C, were not signiﬁcantly aﬀected by either MTSES or MTSET (e.g., Figure 3C), as recently demonstrated for Cys- less using similar protocols28,30 (Figure 4). The shape of the macroscopic I−V relationship was nearly linear for Cys-less CFTR28 and was not signiﬁcantly altered by any of the mutations studied, nor did modiﬁcation by either MTSES or MTSET have any signiﬁcant eﬀect on the shape of the I−V relationship (Figure 3D,E and Figure S1 of the Supporting Information). The eﬀects of MTS reagents on the amplitude of macroscopic currents carried by diﬀerent channel constructs are summarized in Figure 4. The rate of modiﬁcation of introduced cysteines was quantiﬁed from the time course of macroscopic current amplitude change following application of MTSES or MTSET (Figure 5). Eﬀects of MTSES on Single-Channel Currents. To further characterize the nature of the inhibitory eﬀects of MTS reagents on cysteine-substituted CL3 mutants, we recorded single-channel currents carried by these mutants in inside-out patches. For technical reasons, single-channel recordings were taken using a low extracellular Cl− concentration of 4 mM (see Materials and Methods). Under these conditions, all channel constructs studied had identical single-channel conductance, even where the mutation involved removal of a positive charge (K946C and R975C) (Figure 6). Note that those six mutants that failed to generate macroscopic currents (see above) were also not associated with detectable single-channel currents. We restricted our single-channel studies to the use of MTSES, because this reagent had more consistent eﬀects on cysteine-substituted CL3 mutants [i.e., all MTS-sensitive mutants were inhibited by MTSES, although not all were inhibited by MTSET (Figure 4)] and also because we considered modiﬁcation by this negatively charged substance more likely to interfere electrostatically with Cl− permeation and so more likely to inﬂuence Cl− conductance.28−30 In fact, in most cases, MTSES and MTSET had similar eﬀects on macroscopic current amplitudes (Figure 4). The approach we followed is illustrated in Figure 7, using I982C as an example. PKA- and ATP-stimulated single-channel currents were recorded for >5 min to ensure stable activation, following which MTSES was applied to the intracellular solution. In the example shown in Figure 7, it can be seen that the application of MTSES reduced the open probability of the channel to ∼55% of the control, without aﬀecting the single-channel current amplitude. Eﬀects of MTSES on both unitary current amplitude and open probability were quantiﬁed using all-points amplitude histograms constructed from data before and after MTSES exposure (e.g., Figure 7B). This data for all channel 3974 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981