ABCMdb

PMID: 20805575

Bai Y, Li M, Hwang TC
Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
J Gen Physiol. 2010 Sep;136(3):293-309., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCC7 p.Met348Cys ABCC7 p.Ile344Cys For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. Login to comment 18 ABCC7 p.Arg352Cys For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. Login to comment 30 ABCC7 p.Val510Ala Subsequently, an additional V510A mutation was introduced to promote expression (Wang et al., 2007). Login to comment 31 ABCC7 p.Val510Ala A single cysteine was then engineered into each position in TM6 on the cysless/V510A background using the QuikChange XL kit (Agilent Technologies). Login to comment 33 ABCC7 p.Val510Ala Because all of our studies were performed under this cysless/ V510A background, we will refer to this construct as our wild-type (WT) cysless control. Login to comment 82 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment 83 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Ser341Cys The single-channel amplitude is unsolv- able in the cases of R334C, S341C, R347C, and R352C due to a limited bandwidth, whereas it is 0.2-0.3 pA for Data analysis Current traces containing fewer than three channel opening levels and lasting for >1 min were selected for single-channel kinetic analysis using a program developed by L. Csanády (2000). Login to comment 95 ABCC7 p.Phe337Cys Single-channel amplitude: cysless/WT,0.46±0.005pA(n=5); cysless/K355C, 0.28 ± 0.011 pA (n = 4); cysless/F337C, 0.19 ± 0.008 pA (n = 3). Login to comment 107 ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Met348Cys ABCC7 p.Ile344Cys Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment 120 ABCC7 p.Met348Cys In addition, MTSET modification can more than double the current in M348C channels. Login to comment 123 ABCC7 p.Met348Cys A representative result of the experiments with the cysless/ M348C construct is shown in Fig. 5. Login to comment 128 ABCC7 p.Ser341Cys Although MTSET posed small (<20% for S341C) or negligible inhibition, modification by MTSES drastically reduced the current (e.g., Fig. 4, E and F). Login to comment 133 ABCC7 p.Thr351Cys (A and B) Neither MTSES nor MTSET altered the current of cysless/T351C channels. Login to comment 135 ABCC7 p.Met348Cys (D) MTSET increased ATP-dependent current in cysless/M348C channels. Login to comment 137 ABCC7 p.Gln353Cys (E) MTSES reduced the current of cysless/Q353C channels. Login to comment 138 ABCC7 p.Gln353Cys (F) For cysless/Q353C, the treatment of MTSET had little influence on channel function, but prevented the current decrease in response to MTSES, as in E. behavior and conduction properties were recovered after the application of 10 mM DTT. Login to comment 144 ABCC7 p.Arg352Cys Fig. 7 depicts a representative recording of a patch containing a single cysless/R352C CFTR channel. Login to comment 146 ABCC7 p.Met348Cys Interestingly, MTSET modification of M348C also slightly but significantly increased (12 ± 1%; n = 5) the single-channel amplitude (Fig. 5, A and C). Login to comment 154 ABCC7 p.Met348Cys Again, gating Figure 5. Effects of MTSET on a single cysless/M348C channel. Login to comment 156 ABCC7 p.Met348Cys (B) Gating parameters of the cysless/M348C channel before (black) and after (blue) modification, as extracted from the traces in A. Those of the cysless/ WT (gray) in the presence of 2 mM ATP are also included for comparison (traces not depicted). Login to comment 157 ABCC7 p.Met348Cys n = 5 for cysless/M348C. Login to comment 159 ABCC7 p.Met348Cys (C) Single-channel amplitude of the cysless/M348C channel before and after modification in the same patch. Login to comment 162 ABCC7 p.Arg352Cys The restoration of the single-channel amplitude by MTSET or MTSEA on R352C compelled us to use another strategy to assess the function of the CFTR pore before and after modification. Login to comment 163 ABCC7 p.Arg352Cys Fig. 8 shows results obtained from patches yielding macroscopic cysless/R352C channel currents. Login to comment 167 ABCC7 p.Arg352Cys When the membrane voltage was held at 50 mV, 50 µM glibenclamide induced 27.2 ± 1.7% (n = 10) block of the Cl current for cysless/R352C. Login to comment 171 ABCC7 p.Arg352Cys However, when R352C was modified by MTSET, the single-channel amplitude was restored to that of the cysless/WT channel (Fig. 7 A). Login to comment 173 ABCC7 p.Arg352Cys Interestingly, although bringing back the positive charge at this position with MTSET completely restores the single-channel amplitude, gating of MTSET-modified cysless/ R352C is not fully recovered to the level of cysless/WT channels. Login to comment 179 ABCC7 p.Ile344Cys (B) The Po, obtained from the traces in A, of the cysless/I344C channel before (black) and after (blue) modification. Login to comment 181 ABCC7 p.Ile344Cys n = 7 for cysless/ I344C. Login to comment 183 ABCC7 p.Ile344Cys (C) Single-channel amplitude of the cysless/I344C channel before and after modification. Login to comment 185 ABCC7 p.Arg352Cys ABCC7 p.Ser341Cys Because anion conduction was severely perturbed by the mutations R352C and S341C, we were not able to assess the effects of MTSES modification on the single-channel amplitude for these two constructs. Login to comment 186 ABCC7 p.Gln353Cys ABCC7 p.Met348Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys Instead, we will focus on the four other positive hits (i.e., I344C, V345C, M348C, and Q353C). Login to comment 187 ABCC7 p.Gln353Cys Fig. 9 depicts a sample experiment with the cysless/Q353C construct. Login to comment 190 ABCC7 p.Arg352Cys For the positions 344, 345, and 348, however, single-channel recordings are not helpful because no visible current glibenclamide at the same holding potential induced stronger block in the cysless/WT channel (48.6 ± 3.0%; n = 5; Fig. 8 D), the effect of the blocker was attenuated by the R352C mutation. Login to comment 197 ABCC7 p.Arg352Cys (A; top trace) A continuous recording showing the effects of MTSET or MTSEA on a single cysless/R352C channel. Login to comment 201 ABCC7 p.Arg352Cys ABCC7 p.Met348Cys (B) Single-channel amplitude, Po, open time and closed time of MTSET- (blue) and MTSEA-modified (green) cysless/R352C channel, as determined by Gaussian fitting and kinetics analysis; n = 6. inhibition of the macroscopic mean current (Fig. 4 C) and the single-channel current in the case of the cysless/ M348C channel might be due to oxidation of the introduced cysteine to a state not reactive toward either DTT or MTS reagents. Login to comment 208 ABCC7 p.Ile344Cys In a representative recording of a patch containing hundreds of cysless/I344C channels (Fig. 10), one can clearly discern the single-channel amplitude from the expanded trace before and after phosphorylation-dependent activation. Login to comment 212 ABCC7 p.Met348Cys ABCC7 p.Val345Cys Similar results were obtained with the cysless/V345C and cysless/ M348C channels. Login to comment 214 ABCC7 p.Arg352Cys Although the exact reason is unknown, the discrepancy between the extent of Figure 8. Blocking of cysless/ R352C channels by glibenclamide before and after MTS modification. Login to comment 217 ABCC7 p.Arg352Cys (D) The fraction of block, calculated as (1Ig/ I0) × 100% (Ig and I0 are the mean current in the presence of ATP and ATP plus glibenclamide, respectively), for cysless/WT channels (gray), cysless/R352C channels before modification (black) and after modification with MTSET (blue), and after modification with MTSEA (green). Login to comment 220 ABCC7 p.Gln353Cys This representative recording (among five patches) contains two cysless/ Q353C channels. Login to comment 235 ABCC7 p.Arg352Cys The reason for this slow reaction rate is unclear, but it could be due to a perturbation of the pore architecture by the R352C mutation (Cui et al., 2008). Login to comment 240 ABCC7 p.Val345Cys A representative experimental result with the cysless/V345C construct is shown in Fig. 11 A. Login to comment 241 ABCC7 p.Ile344Cys The modification rate by 1 mM MTSES when Figure 10. MTSES decreased single-channel amplitude of cysless/I344C channels. Login to comment 256 ABCC7 p.Met348Cys ABCC7 p.Val345Cys (A and B) Macroscopic recordings of cysless/ V345C and cysless/M348C showing modification by 1 mM MTSES when the membrane potential is held at 50 mV (left) and 100 mV (right). Login to comment 274 ABCC7 p.Ile344Cys (B) A continuous single-channel recording of cysless/I344C showing a dramatic increase of the spontaneous ATP-independent gating after MTSET modification. Login to comment 279 ABCC7 p.Met348Cys Similar results were obtained for cysless/M348C: 0.36 ± 0.03 s (n = 3) before and 0.55 ± 0.03 s (n = 3) after modification. Login to comment 290 ABCC7 p.Thr351Cys ABCC7 p.Thr1142Cys ABCC7 p.Met348Cys For example, biochemical studies demonstrated that both M348C and T351C can be cross-linked to T1142C in TM12 (Chen et al. 2004). Login to comment 302 ABCC7 p.Arg352Cys Second, the potency to an open-channel blocker, glibenclamide, is reduced by the charge-neutralizing mutation R352C (also see Cui et al., 2008), but was restored by charge-restoring adducts. Login to comment 314 ABCC7 p.Ile344Cys However, our results showing drastic alterations in gating kinetics caused by modulating TM6 in the TMDs (e.g., a long-lasting opening on the order of tens of seconds with the MTSET-modified cysless/I344C channel) raise the possibility that gating motion in the TMDs can also affect ATP binding and hydrolysis in the NBDs (compare Kogan et al. 2001), a subject worth more extensive future explorations. Login to comment 316 ABCC7 p.Met348Cys ABCC7 p.Ile344Cys In fact, our data show that the Po of spontaneous gating in the absence of ATP (Bompadre et al., 2007; Wang et al., 2010) is visibly increased by MTSET modification (Figs. 4 D and 12 B; Po is 0.19 ± 0.04, n = 5 for cysless/M348C, and 0.63 ± 0.03, n = 4 for cysless/ I344C). Login to comment 317 ABCC7 p.Ile344Cys The single-channel trace of cysless/I344C in Fig. 12 B illustrates a negligible ATP-independent gating before MTSET modification, whereas frequent opening and closing events in the absence of ATP can Locher, 2006) as a template. Login to comment 345 ABCC7 p.Arg347Cys Indeed, in the current study, R347C is insensitive to either MTSET or MTSES. Login to comment 420 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment