ABCMdb

PMID: 22042986

Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PubMed]

Sentences

No. Mutations Sentence Comment

62 ABCC7 p.Thr1142Cys Fig. S2 demonstrates that the positively charged 2-trimethylaminoethyl MTS (MTSET+ ) adduct in TM12 enhances the effect of an anionic blocker, glibenclamide. Fig. S3 is a representative trace that illustrates protection of a substituted cysteine in TM12 by glibenclamide. Fig. S4 demonstrates modification of cysless/T1142C channels in the presence of ATP plus pyrophosphate. Login to comment 63 ABCC7 p.Glu1371Gln Fig. S5 shows the data for modification rates in the cysless/ E1371Q background. Login to comment 64 ABCC7 p.Trp1145Cys Fig. S6 depicts a representative trace showing modification of cysless/W1145C channels in the absence of ATP. Login to comment 114 ABCC7 p.Ser1141Cys (A) Current recorded at 50 mV in inside-out membrane patches containing thousands of cysless/S1141C-CFTR channels. Login to comment 118 ABCC7 p.Ile1139Cys (B) Representative current recording of cysless/ I1139C-CFTR channels using a similar experimental protocol as in A. Login to comment 133 ABCC7 p.Ser1141Cys Consistent with this idea, the S1141C-CFTR channel, when modified by 2-aminocarbonylethyl MTS (MTSACE), a similarly sized but neutral reagent, also displayed a smaller single-channel current amplitude (Fig. 3 D). Login to comment 137 ABCC7 p.Ser1141Cys (A and B) Measurements of the second-order reaction rate constant (MTSES  ) of MTSES modification in the presence of 2 mM ATP for cysless/Q1144C(A)andcysless/S1141C (B). Login to comment 154 ABCC7 p.Trp1145Cys ABCC7 p.Asn1148Cys (A and B) Single-channel recordings at 50 mV show that application of MTSET+ increases unitary current amplitudes for cysless/N1148C (A) or cysless/W1145C (B). Login to comment 155 ABCC7 p.Trp1145Cys ABCC7 p.Asn1148Cys Linear fits to single-channel current measurements at different voltages yield unitary conductance values for cysless/ N1148C (A) of 7.1 pS before (black) and 9.2 pS after (blue) MTSET+ treatment, for cysless/W1145C (B) of 7.6 pS before (black) and 10.1 pS after (blue) MTSET+ treatment. Login to comment 156 ABCC7 p.Ser1141Cys (C and D) Single-channel recordings show that the unitary current amplitude decreases after MTSET+ (C; blue) or MTSACE (D; green) exposure for cysless/S1141C. Login to comment 157 ABCC7 p.Ser1141Cys Linear fits to single-channel current measurements at different voltages yield unitary conductance values for cysless/S1141C of 7.0 pS before (C and D; black) and 6.1 pS after both MTSET+ (C; blue) and MTSACE (D; green) treatment. Login to comment 159 ABCC7 p.Trp1145Cys (A) In an inside-out patch containing thousands of cysless/W1145C-CFTR channels, MTSES was first (indicated by orange lines) applied in the presence of 200 µM glibenclamide (yielding 77% steady-state blockade), and then (red lines) in the absence of glibenclamide after the chemical modification was reversed by the reducing reagent, DTT. Login to comment 180 ABCC7 p.Ser1141Cys ABCC7 p.Asn1148Cys Note that the modification is faster in the presence of ATP than in the absence of ATP for cysless/N1148C-CFTR channels (B), whereas it is slower in the presence of ATP than in the absence of ATP for cysless/S1141C-CFTR channels (C). Login to comment 194 ABCC7 p.Ser1141Cys (A) Chemical modification of cysless/S1141C-CFTR channels by Texas red MTSEA+ (orange arrows) without ATP. Login to comment 198 ABCC7 p.Met348Cys ABCC7 p.Ile344Cys ABCC7 p.Ser1141Cys ABCC7 p.Asn1148Cys (C) Second-order rate constants (MTSES  ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels. Login to comment 203 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Given that the E1371Q mutant channel has an open probability of near unity in the presence of ATP, this result suggests that 1141C is readily accessible to MTSES but not to Texas red MTSEA+ in an open channel. Similar results were observed for cysteines substituted into positions 1148, 344, and 348 under the E1371Q background. Login to comment 209 ABCC7 p.Asn1138Cys ABCC7 p.Ser1149Cys However, although they detected reactivity in N1138C and S1149C, we found that these sites appear nonreactive. Login to comment 220 ABCC7 p.Thr1142Cys ABCC7 p.Thr1142Cys Because the modification rate of T1142C in Linsdell`s study is >60-fold faster than that of ours, we measured the modification rate of T1142C in the presence of ATP plus pyrophosphate and found that the modification rate is not significantly different from that measured with ATP alone (Fig. S4). Login to comment 224 ABCC7 p.Glu1371Gln To better assess the modification rate of an open channel, we introduced an additional E1371Q mutation, which abolishes ATP hydrolysis and thereby confers an open probability of 1 on the CFTR channel. Login to comment 225 ABCC7 p.Glu1371Gln Under the E1371Q background, MTSES readily modified 1141C resulting in a decrease of current (Fig. 6 D, red line), but Texas red MTSEA+ did not react with 1141C because it neither substantially decreased the macroscopic current (Fig. 6 D, orange line) nor prevented 1141C from subsequent modification by MTSES . Login to comment