Biochemistry addition to its severe traﬃcking defect,34,35 Cys-less CFTR has been reported to have gating31,36 and permeation31,36,37 properties subtly diﬀerent from those of wild-type CFTR. Additional mutations were introduced into the Cys-less background using the QuikChange site-directed mutagenesis system (Agilent Technologies, Santa Clara, CA) and veriﬁed by DNA sequencing. Mutations were introduced into the juxtamembrane regions of CL3, replacing each consecutive amino acid between L941 and K951 (in the cytoplasmic extension from TM8) and from L973 to L983 (in the cytoplasmic extension from TM9) individually with cysteine (Figure 1E). BHK cells were transiently transfected as described previously,38 except that 24 h after transfection cells were transferred to 27 °C to promote mature protein expression.32 Cells were used for Western blotting analysis after 48 h at 27 °C, and for electrophysiological experimentation after 1−3 days at 27 °C. For Western blotting, cells at ∼80% conﬂuence were washed twice with ice-cold PBS, harvested by being scraped, and lysed on ice for 30 min in RIPA buﬀer supplemented with protease inhibitors. Cells were lysed by repeated vortexing as well as by passing the lysate through a pipet tip every 5 min. The lysate was then centrifuged (15000g for 30 min at 4 °C) and the supernatant poured into a fresh tube. The protein concen- tration was assayed using the Bradford protein assay method. Approximately 30 μg of protein was preincubated with an equal volume of loading buﬀer for 30 min at 37 °C and then loaded onto a 7.5% acrylamide−SDS gel and ﬁnally transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Immuno- blotting for CFTR was performed by incubating the membrane overnight at 4 °C with monoclonal mouse anti-CFTR antibody (M3A7, Millipore) at a 1:1000 dilution. After being washed, the membrane was incubated for 1 h at room temperature with a secondary antibody (horseradish peroxidase-conjugated goat anti-mouse, Jackson ImmunoResearch, West Grove, PA) at a 1:5000 dilution. Detection was conducted using the ECL Plus kit (Amersham Pharmacia, Baie d’Urfe, QC) following the manufacturer’s instructions. The relative expression of mature, complex glycosylated CFTR protein (“Band C”) and immature, core glycosylated protein (“Band B”) was assessed by densitometric analysis of scanned Western blots using 1-D (Bio-Rad, Hercules, CA). Macroscopic and single-channel recordings were made using the excised, inside-out conﬁguration of the patch clamp technique. CFTR channels were activated after patch excision and recording of background currents by exposure to protein kinase A catalytic subunit (PKA, 20 nM) and MgATP (1 mM) in the cytoplasmic solution. For macroscopic current record- ings, both the intracellular (bath) and extracellular (pipet) solution contained 150 mM NaCl, 10 mM N-tris- (hydroxymethyl)methyl-2-aminoethanesulfonate, and 2 mM MgCl2. For single-channel recordings, NaCl in the pipet solution was replaced with 150 mM sodium gluconate to generate an outwardly directed Cl− concentration gradient. This was done to allow CFTR single-channel currents to be resolved at a membrane potential of −20 mV during the prolonged recordings necessary for measurement of channel open probability (see Figure 7). During preliminary recordings, we found that inside-out patches held at more negative or positive membrane potentials using the same symmetrical Cl− solutions used for macroscopic current recordings were unstable over these prolonged recording periods. The trans- membrane Cl− concentration gradient is not expected to Article inﬂuence any of the parameters measured in our experiments. All experimental solutions were adjusted to pH 7.4. Channels were exposed to intracellular cysteine-reactive MTS reagents to covalently modify an introduced cysteine side chain. Two MTS reagents, the negatively charged [2- sulfonatoethyl] MTS (MTSES) and the positively charged [2-(trimethylammonium)ethyl] MTS (MTSET) were used at high concentrations (200 μM) that had no eﬀect on Cys-less CFTR.28,32 MTS reagents were applied to the cytoplasmic face of inside-out patches after stable current activation with PKA and ATP, and the current was monitored for at least 5 min to ensure stable modiﬁcation. In some cases, the identity of the remaining currents as being carried by CFTR was conﬁrmed using the CFTR-speciﬁc inhibitor CFTRinh-172. To measure the rate of modiﬁcation, the macroscopic current amplitude was monitored continuously and the time-dependent change in amplitude after the addition of MTS reagent was ﬁtted by a single-exponential function. The time constant of exponential current decay, τ, was then used to calculate the apparent second-order reaction rate constant, k, from the equation k = 1/ ([MTS]τ). Current traces were ﬁltered at 100 Hz (macroscopic currents) or 50 Hz (single-channel currents) using an eight- pole Bessel ﬁlter, digitized at 250 Hz, and analyzed using pCLAMP9 (Molecular Devices, Sunnyvale, CA). Macroscopic current−voltage (I−V) relationships were constructed using voltage ramp protocols from a holding potential of 0 mV. The single-channel current amplitude and channel open probability were estimated from all-points amplitude histograms (Figure 7). For analysis of the eﬀects of MTSES on single-channel currents, currents were recorded for at least 5 min at a membrane potential of −20 mV under control conditions to ensure stable channel activity, following which MTSES was applied to the cytoplasmic face of the patch and currents were recorded for a further 5−12 min. Because channel open probability was highly variable between patches (see Figure S2 of the Supporting Information), we have focused on the eﬀect of MTSES exposure on open probability, which we found to be considerably more consistent (Figure 8B). Experiments were conducted at room temperature (21−24 °C). Values are presented as means ± the standard error of the mean. Tests of signiﬁcance were conducted using an unpaired t test, for which p < 0.05 was considered statistically signiﬁcant. All chemicals were obtained from Sigma-Aldrich (Oakville, ON), except MTSES and MTSET (Toronto Research Chemicals, North York, ON) and PKA (Promega, Madison, WI). ■ RESULTS Expression of CL3 Mutants in a Cys-less CFTR Background. We used site-directed mutagenesis to introduce cysteines into the juxtamembrane region of CL3 (Figure 1). Cysteines were substituted for each amino acid from L941 to K951 in the cytoplasmic extension from TM8, and from L973 to L983 in the extension from TM9 (Figure 1E). Mutant CFTR was expressed in BHK cells, and proper processing of the CFTR protein was assayed using Western blot analysis. As shown in Figure 2, Cys-less CFTR was able to generate mature, complex glycosylated (Band C) protein, although the relative abundance of this mature form was lower than that of wild-type CFTR. Previously, we have shown that the ability of Cys-less to produce Band C protein in BHK cells is dependent on (i) the growth of the cells at a reduced temperature of 27 °C and (ii) 3973 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981