ABCMdb

PMID: 15504728

Zhang ZR, Cui G, Liu X, Song B, Dawson DC, McCarty NA
Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore.
J Biol Chem. 2005 Jan 7;280(1):458-68. Epub 2004 Oct 25., 2005-01-07 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Arg334Cys Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. Login to comment 5 ABCC7 p.Arg334Cys The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. Login to comment 20 ABCC7 p.Arg334Cys EXPERIMENTAL PROCEDURES Preparation of Oocytes and cRNA-For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers (14). Login to comment 21 ABCC7 p.Arg334Cys R334C was prepared from a construct carrying the full coding region of CFTR in the pBluescript vector. Login to comment 43 ABCC7 p.Arg334Cys Since preliminary experiments showed that the full single channel conductance of unmodified R334C-CFTR was very low (1.5 pS) compared with that of WT-CFTR (14, 15), most single-channel experiments in this study used asymmetrical [Cl- ] in order to increase the single channel amplitude at VM ϭ -100 mV. Login to comment 64 ABCC7 p.Arg334Cys Analysis of Single Channel and Macropatch Experiments-The Fetchan and pSTAT programs of pClamp 8.0 were used to calculate open probability (Po) and to make all-points amplitude histograms for R334C-CFTR channels before and after modification. Login to comment 71 ABCC7 p.Arg334Cys As described below, R334C channels exhibit multiple conductance levels, with s1 representing subconductance level 1, s2 being subconductance level 2, and f being full conductance level, as well as the closed level (c). Login to comment 75 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys It is noteworthy that because the single channel amplitude of R334C is very small, we transferred the all-points histogram data for R334C-CFTR channels to PeakFit version 4.11 (SYSTAT Software Inc., Chicago, IL) to verify the result of fits in pClamp 8.0. Login to comment 93 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys Fig. 1 also contains records illustrating the subconductance behavior of two mutant CFTRs: R334C and T338A. Login to comment 94 ABCC7 p.Thr338Ala The former exhibits a full conductance that is less than that of WT-CFTR under comparable conditions (14), and the latter exhibits an increased full conductance (9.8 pS in T338A-CFTR). Login to comment 97 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys This result suggests that neither the R334C nor T338A mutation, although they involve residues reputed to lie within the CFTR pore (14, 18), greatly altered the relative magnitude of the subconductance states. Login to comment 99 ABCC7 p.Arg334Cys In a longer record of single-channel currents from oocytes expressing R334C-CFTR (Fig. 2, A and B), the most prominent state is discernible as a conductance of ϳ1.2 pS that was reported previously (14), although upon closer examination of the fine structure of the open bursts, it is found that within nearly every burst there are transitions to three conductance states: one lower in conductance than that of the most frequent state and one higher in conductance. Login to comment 101 ABCC7 p.Arg334Cys Deposition of a Positive Charge at 334 Amplifies All Conductance States Proportionately-We reasoned that if the three conductance states reflect different behaviors of a shared portion of the conduction path, which includes the amino acid at position 334 in TM6, it would be possible to use the properties of the R334C mutant to investigate the architecture of the functional CFTR pore. Login to comment 102 ABCC7 p.Arg334Cys We showed previously that covalent modification of R334C-CFTR channels with MTSETϩ increased the amplitude of the most prominent single-channel conductance (referred to here as s2) without altering gating (14). Login to comment 107 ABCC7 p.Arg334Cys R334C-CFTR chloride channels were modified in ϳ15 min by MTSETϩ diffusing down the electrode tip, as reflected by an increment of s1, s2, and f conductance levels ϳ2.12.3-fold (Fig. 2, B-F). Login to comment 108 ABCC7 p.Arg334Cys We analyzed eight paired, inside-out single channel records (both pre-and postmodified channels included in the same patch) that contained only one R334C-CFTR channel per patch, as shown in Fig. 2A. Login to comment 111 ABCC7 p.Arg334Cys We maintained the patches that contained modified R334C-CFTR channels for up to 45 min in some experiments and found that following modification by MTSETϩ , the amplitudes of the s1, s2, and f conductance states consistently stayed at the same level, with no further modification observed. Login to comment 115 ABCC7 p.Arg334Cys Hence, the shared impact of covalent modification by MTSETϩ on the amplitude of all conductance states exhibited by R334C-CFTR channels was also consistent with the hypothesis that the three conductance states have at least a portion of the conduction path in common. Login to comment 116 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Covalent Modification of R334C-CFTR Did Not Alter Gating-We previously reported that modification of R334C-CFTR by MTSETϩ did not alter gating as defined by comparing open probability in patches from treated and untreated oocytes as well as that of channels monitored during the process of modification (14). Login to comment 117 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Analysis of the subconductance behavior of R334C-CFTR provided an opportunity to reexamine the question of possible gating effects by asking whether modification of R334C-CFTR channels by MTSETϩ altered the prevalence or duration of the three conductance states. Login to comment 119 ABCC7 p.Arg334Cys The overall Po of R334C-CFTR channels before and after MTSETϩ modification was 0.24 Ϯ 0.04 and 0.23 Ϯ 0.04, respectively (p ϭ 0.91; see Fig. 3D). Login to comment 120 ABCC7 p.Arg334Cys Furthermore, as shown in Fig. 2G, the fractional abundance of the s1, s2, and f conductance states did not change upon MTSETϩ -induced modification in R334C-CFTR channels. Login to comment 124 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys A-C, records for WT-, R334C-, and T338A-CFTR, respectively, were generated in excised, inside-out mode with asymmetrical [Cl- ], where the pipette was filled with 40 mM [Cl- ] and bath (cytoplasmic) solution contained 302 mM [Cl- ] in order to potentiate the single channel amplitude. Login to comment 128 ABCC7 p.Arg334Cys Furthermore, the observation in R334C-CFTR that the amplitudes of the s1, s2, and f states increased by an equivalent proportion, and apparently simultaneously, upon modification by MTSETϩ suggests that each of these states reflects the activity of a single pore, or a portion of a shared conduction pathway rather than the activity of two separate pores. Login to comment 130 ABCC7 p.Arg334Cys Modification of R334C-CFTR increases the conductance of each open state. Login to comment 131 ABCC7 p.Arg334Cys A, representative trace from an oocyte expressing R334C-CFTR in the detached, inside-out patch configuration during real time modification; VM ϭ -100 mV, with asymmetrical [Cl- ]. Login to comment 136 ABCC7 p.Arg334Cys B and C, two isolated single bursts representing R334C-CFTR from the same patch before (B) and after (C) modification by MTSETϩ . Login to comment 156 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys A similar result was obtained using a double mutant, R334C/K1250A, that exhibits a prolonged open state duration (data not shown). Login to comment 163 ABCC7 p.Arg334Cys In Fig. 4, activated R334C-CFTR channels were first exposed to the bath solution containing no MTSETϩ for a time period of ϳ20 s and then perfused by bath solution containing 50 ␮M MTSETϩ . Login to comment 164 ABCC7 p.Arg334Cys R334C-CFTR macroscopic current increased rapidly, reflecting modification by MTSETϩ (14). Login to comment 165 ABCC7 p.Arg334Cys The final, steady-state macroscopic current amplitude of modified R334C-CFTR was increased by 2.3 Ϯ 0.22-fold after prolonged exposure to 50 ␮M MTSETϩ . Login to comment 171 ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys This observation provided an opportunity to test directly the notion that a nearby positive charge would modify the rate of modification of the cysteine at 334 by comparing the rate of modification of R334C-CFTR with the rate of modification of R334C/K335A-CFTR (Fig. 4B). Login to comment 172 ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys The amplitude of macroscopic current was increased 2.97 Ϯ 0.24-fold by 50 ␮M MTSETϩ in R334C/K335A-CFTR. Login to comment 174 ABCC7 p.Lys335Ala These data are compatible with the notion that the rate of modification of a cysteine at position 334 is sensitive to the local electrostatic potential, partially determined by the amino acid at position 335, although we cannot discount the possibility that the K335A mutation altered the pore structure FIG. 3. Login to comment 175 ABCC7 p.Arg334Cys Modification of R334C-CFTR does not affect reversal potential and open probability. Login to comment 177 ABCC7 p.Arg334Cys D, Po determined before (filled circles) and after (filled triangles) covalent modification by MTSETϩ for eight paired patches containing single R334C-CFTR channels. Login to comment 180 ABCC7 p.Arg334Cys in the vicinity of R334C. Login to comment 182 ABCC7 p.Arg334Cys As an additional test for the presence of multiple cysteines, we studied the kinetics of modification of R334C-CFTR channels in outside-out macropatches using a two-pulse protocol as follows. Login to comment 189 ABCC7 p.Arg334Cys If there were two cysteines in each one-pore CFTR, then following the first brief exposure to MTSETϩ at a low concentration, the pool of R334C-CFTR channels should comprise a mixed population of unmodified, singly modified, and doubly modified channels (Fig. 5C); longer exposure to MTSETϩ in the first treatment would lead to a greater increase in current, due to modification of more cysteines. Login to comment 190 ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys The change in electrostatic potential due to modification of one cysteine would be expected to alter the rate of modification of the remaining cysteine, as suggested by the difference in response in R334C- and R334C/ K335A-CFTR. Login to comment 194 ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Outside-out macropatch experiments for mutants R334C- and R334C/K335A-CFTR. Login to comment 197 ABCC7 p.Arg334Cys A, an example of macroscopic current of R334C-CFTR, filtered at 200 Hz. Login to comment 199 ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys The amplitude of macroscopic current was increased by 2.3-fold upon modification by MTSETϩ in this experiment. B, representative macroscopic current of R334C/K335A-CFTR; the red line is the curve fit, with ␶ ϭ 1.1 s in this experiment. Login to comment 201 ABCC7 p.Arg334Cys R334C-CFTR channels have one engineered cysteine per pore. Login to comment 202 ABCC7 p.Arg334Cys A, outside-out macropatch experiment for R334C-CFTR, using a protocol similar to that shown in Fig. 4 but with two exposures to MTSETϩ . Login to comment 206 ABCC7 p.Arg334Cys In experiments such as that shown in Fig. 5A, brief exposure to 5-10 ␮M MTSETϩ should modify a subset of the available cysteines, resulting in one of three conditions: (i) in some R334C-CFTR pores, neither of the cysteines would be modified; (ii) in some pores, only one cysteine would be modified; and (iii) in some pores, both of the two cysteines within a single pore would be modified (stars indicate the cysteines that were modified by MTSETϩ ). Login to comment 213 ABCC7 p.Arg334Cys To determine whether any engineered cysteines remain unmodified in R334C-CFTR channels after prolonged exposure to MTSETϩ , we took advantage of the sensitivity of unmodified cysteines to bath pH. Login to comment 214 ABCC7 p.Arg334Cys R334C-CFTR channels were examined by two-electrode voltage clamp, and channels were activated via the beta2-adrenergic receptor by exposure to isoproterenol. Login to comment 215 ABCC7 p.Arg334Cys As reported previously, the conductances of oocytes expressing unmodified R334C-CFTR channels were sensitive to bath pH, due to titration of the partial negative charge on the unmodified cysteine (14). Login to comment 217 ABCC7 p.Arg334Cys The macroscopic conductance of R334C-CFTR was increased ϳ2.5-fold (n ϭ 3, Fig. 6B) upon MTSETϩ -induced modification at bath pH 7.5, which is consistent with our previous report (14). Login to comment 218 ABCC7 p.Arg334Cys However, after R334C-CFTR channels were covalently modified by 200 ␮M MTSETϩ , the macroscopic conductance was no longer sensitive to pH titration (Fig. 6, B and D). Login to comment 221 ABCC7 p.Arg334Cys The possibility remains, however, that two separate copies of R334C contribute to each functional pore and that MTSETϩ - induced modification of these two targets occurs with identical rates as expected if the two sites are far enough apart in the folded channel polypeptide that the electrostatic charge change that accompanies modification of one site is not sensed at the other site. Login to comment 224 ABCC7 p.Arg334Cys Although our previous experiments showed that the number of R334C-CFTR channels resident at the oocyte plasma mem- FIG. 6. Login to comment 225 ABCC7 p.Arg334Cys R334C-CFTR was not accessible to protons after modification by MTSET؉ . Login to comment 226 ABCC7 p.Arg334Cys A and C, oocytes expressing R334C-CFTR and beta2-adrenergic receptor were first activated by ND96 plus 5 ␮M isoproterenol at pH 7.5 for 6 min. Login to comment 228 ABCC7 p.Arg334Cys On average, the macroscopic conductance of R334C-CFTR increased 22 Ϯ 3% (p ϭ 0.02, n ϭ 3) in pH 6.0 and decreased 50 Ϯ 5% (p ϭ 0.01, n ϭ 3) in pH 9.0. Login to comment 229 ABCC7 p.Arg334Cys B and D, oocytes expressing R334C-CFTR and beta2-adrenergic receptor were first activated by ND96 plus 5 ␮M isoproterenol at pH 7.5 for 6 min and then followed by the same solution containing 200 ␮M MTSETϩ for 4 min; the macroscopic conductance was increased by ϳ2.5-fold upon application of MTSETϩ . Login to comment 230 ABCC7 p.Arg334Cys Modification by MTSETϩ prevented the pH-induced response seen in R334C-CFTR macroscopic conductance. Login to comment 232 ABCC7 p.Arg334Cys To control for potential changes in channel number, we analyzed single-channel recordings containing multiple R334C-CFTR channels in excised mode, while MTSETϩ diffused down to the tip from a back-filled pipette; the example shown in Fig. 7 contained at least three active channels. Login to comment 243 ABCC7 p.Arg334Cys R334C-CFTR channels contain a single population of engineered cysteines. Login to comment 244 ABCC7 p.Arg334Cys A, an individual patch containing at least three R334C-CFTR channels. Login to comment 246 ABCC7 p.Arg334Cys The upper trace includes the first modified R334C-CFTR opening, indicated by the filled arrowhead. Login to comment 247 ABCC7 p.Arg334Cys Through time, the number of unmodified R334C-CFTR openings was reduced (middle trace, indicated by the open arrowheads), and finally all openings exhibited the modified conductance (bottom trace). Login to comment 284 ABCC7 p.Arg334Cys In the present experiments, we compared the subconductance behavior of wild type and mutant CFTRs and investigated the effect on subconductance behavior of covalent charge deposition using R334C-CFTR. Login to comment 291 ABCC7 p.Arg334Cys Neither the impact of covalent labeling nor the kinetics of labeling provided any evidence for the presence of more than a single cysteine in the pore formed by R334C-CFTR. Login to comment 296 ABCC7 p.Arg334Cys First, functional modification of R334C-CFTR by reagents such as MTSETϩ and 2-sulfonatoethyl methanethiosulfonate indicates that a cysteine at position 334 lies within the outward facing, water-accessible surface of the protein. Login to comment