Biochemistry Article Figure 7. Example of the eﬀect of modiﬁcation by MTSES on single-channel currents. (A) Current recorded from a patch containing two I982C channels, before (Control) and after addition of 200 μM MTSES to the intracellular solution, at a membrane potential of −20 mV. The horizontal line to the left of the traces indicates the zero current level. (B) All-points amplitude histograms prepared from extended periods of the recordings shown in panel A, corresponding to control (left) and post-MTSES (right) conditions. Each has been ﬁt by the sum of three Gaussian functions, giving mean unitary amplitudes of −0.504 pA (control) and −0.488 pA (+MTSES) and open probabilities of 0.183 (control) and 0.101 (+MTSES). reasoned that the reduction in macroscopic current amplitude that consistently resulted from MTSES and/or MTSET modiﬁcation of CL3 (Figure 4) could reﬂect a reduction in Cl− conductance (i.e., interference with the movement of Cl− through the open channel pore) and/or a reduction in open probability (i.e., interference with the normal opening and closing of the channel). Direct single-channel recording experiments with MTSES (Figures 7 and 8) support the latter explanation. However, we have additional reasons to doubt an important functional role for CL3 in inﬂuencing the functional properties of the permeation pathway for Cl− ions. We2,28,30 and others3,29 have recently used cytoplasmic MTS reagents to identify pore-lining amino acid side chains in TM1, TM6, and TM12, and several patterns emerge from these studies. In each of these studies, MTSES modiﬁcation inside the pore always causes a reduction in channel function, most likely because of interference with Cl− permeation. This contrasts with MTSES modiﬁcation of CL3, which has no eﬀect on Cl− conductance (Figure 8A). It would be most surprising if introduction of a negative charge by covalent modiﬁcation by MTSES close to the permeation pathway for Cl− ions did not alter Cl− conductance. The rate of MTSES modiﬁcation is high for cysteines introduced close to the cytoplasmic ends of the TMs and decreases with the apparent distance into the pore from its cytoplasmic end, consistent with narrowing of the pore.2,28,30 In contrast, there was no apparent pattern to the rate of MTS modiﬁcation of diﬀerent CL3 residues (Figure 5). Importantly, in TM1 the rate of modiﬁcation by positively charged MTSET is consistently ∼10-fold lower than that by negatively charged MTSES, which was interpreted as reﬂecting the function of the CFTR channel as an anion-selective pore.30 In contrast, although some diﬀerences in the rate of MTSES versus MTSET modiﬁcation were observed for some CL3 residues (Figure 5), there was no consistent pattern suggestive of an overall anion selectivity of this region of CFTR. Instead, we suggest that local factors may result in the small diﬀerences in the rate of MTSES versus MTSET modiﬁcation measured at speciﬁc CL3 residues (Figure 5). The eﬀects of cysteine substitutions per se also do not support an important role for the juxtamembrane regions of CL3 in the Cl− permeation pathway. None of the mutants studied altered the shape of the macroscopic I−V curve (Figure S1 of the Supporting Information) or the single-channel conductance (Figure 6), even though both of these parameters are highly sensitive to cysteine substitutions both in the TMs28,29,33,39−42 and in the ELs that are thought to contribute to the outer mouth of the pore.4,5 The shapes of the I−V relationships were also not altered by MTSES or MTSET modiﬁcation (Figure S1 of the Supporting Information), even though the charge deposition associated with MTS modiﬁcation often aﬀects the rectiﬁcation of the I−V relationship for cysteines located close to the Cl− permeation pathway.4,5,28,33,41,42 Overall, our ﬁndings suggest that, even though molecular models of CFTR suggest that the juxtamembrane region of CL3 might contribute to a restricted region of a central “pore” through the MSDs (Figure 1), this region does not make a suﬃciently important functional contribution to the channel to signiﬁcantly inﬂuence Cl− permeation. One possibility is that CL3 does line the aqueous lumen of the pore, but that Cl− ions do not approach close enough to CL3 for their movement to be inﬂuenced by the properties of individual amino acid side chains here. This would imply that the open pore is very wide at the level of the CLs, much wider than in the TMs where Cl− ions certainly do interact with pore-lining side chains. This suggestion would be consistent with longstanding functional data suggesting the pore has a deep and wide inner vestibule that is readily accessible to large cytoplasmic substances in open channels.1,43 However, it is in direct contrast with the idea, 3978 dx.doi.org/10.1021/bi300065z | Biochemistry 2012, 51, 3971−3981