ABCMdb

PMID: 12172647

Chen JH, Chang XB, Aleksandrov AA, Riordan JR
CFTR is a monomer: biochemical and functional evidence.
J Membr Biol. 2002 Jul 1;188(1):55-71., 2002-07-01 [PubMed]

Sentences

No. Mutations Sentence Comment

54 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala Missense mutations S341A and R347D were generated by site-directed mutagenesis (Stratagene) and cloned into CFTR-M2 by replacing the A¯II-HpaI fragment, and into M2-CFTR by replacing the XbaI-HpaI fragment. Login to comment 79 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala To obtain approximately equal expression of dierent epitope-tagged CFTR-conduction variants, BHK cells were transiently cotransfected with cDNA in the following ratios: 6:1, S341A-M2:WT-HSV;7:5,R347D-M2:WT-HSV;1:1,S341A-M2:TT338, 339AA-HSV;3:11,R347D-M2:TT338,339AA-HSV;6:1,M2-S341A: HSV-WT; and 1:1,M2-R347D:HSV-WT. Login to comment 180 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala Mutations S341A and R347D (single-letter amino acid) dramatically lower chloride conductance (Tabcharani et al., 1993; McDonough et al., 1994) while the double mutation TT338, 339AA enhances chloride conduction (Linsdell et al., 1997). Login to comment 193 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala To the C-terminal ends of CFTR, we attached the M2 epitope to S341A and R347D and the HSV epitope to wild type and TT338, 339AA (S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV, respectively). Login to comment 194 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala Single-channel chord conductances for S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV at À100 mV were (in pS): 2.2, 5.1, 14.3, and 15.6, respectively (Figs. Login to comment 205 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala (C) Amplitude histograms and single-channel recordings of low-conduction mutants S341A and R347D C-terminally tagged with M2 (S341A-M2 and R347D-M2, respectively), and high-conduction variants WT and TT338, 339AA C-terminally tagged with HSV (WT-HSV and TT338, 339AA-HSV, respectively). Login to comment 207 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala (D) Single-channel current-voltage relationships of S341A-M2, R347D-M2, WT-HSV, and TT338,339A-HSV. Login to comment 210 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala (E) Tabulation of single-channel conductances from microsomes containing a low-conducting (S341A.M2 or R347D-M2) and a high-conducting species (WT-HSV or TT338,339AA-HSV). Login to comment 215 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala To assess CFTR stoichiometry, we co-expressed approximately equal amounts of a low-(S341A-M2 or R347D-M2) and a high-conduction species (WT-HSV or TT338,339AA-HSV) for single-channel analysis (Fig. 4E). Login to comment 223 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala (C) Amplitude histograms and single-channel recordings of S341A and R347D N-terminally tagged with M2 (M2-S341A and M2-R347D, respectively), and WT N-terminally tagged with HSV (HSV-WT). Login to comment 225 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala (D) Single-channel current-voltage relationships of M2-S341A, M2-R347D, and HSV-WT. Login to comment 227 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala Intermediate conducting channels were infrequently observed; membrane vesicles containing WT-HSV plus S341A-M2 or R347D-M2 produced 9.5 pS and 7.5 pS channels, and vesicles containing TT338,339AA-HSV plus S341A-M2 or R347D-M2 yielded 12 pS, 8 pS, and 2 pS channels. Login to comment 228 ABCC7 p.Arg347Asp These channels were most likely subconductances of WT-HSV (9.5 pS and 7.5 pS channels), TT338,339AA-HSV (12 pS and 8 pS), and R347D-M2 (2 pS) since they were seen with microsomes containing each of these species alone. Login to comment 235 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala The chord conductances of M2-S341A, M2-R347D, and HSV-WT at À100 mV (in pS) were 2.2, 5.1, and 14.6, respectively (Fig. 5C and 5D). Login to comment 236 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala When co-expressed, microsomes containing HSV-WT and either M2-S341A or M2-R347D produced conductances that were predominantly channels of each constituent in their main conductance states and infrequently their subconductance states (Fig. 5E). Login to comment 237 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala The possibility that these intermediate conductances resulted from a hybrid assembly of multiple CFTR molecules was £ 3.3% and £ 2.1% for HSV-WT plus M2-S341A and M2-R347D, respectively. Login to comment 244 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala (E) Tabulation of single-channel conductances from microsomes containing HSV-WT and either M2-S341A or M2-R347D. Login to comment 247 ABCC7 p.Arg347Asp ABCC7 p.Ser341Ala Co-expressed CFTR proteins do not form a hybrid channel (95% CI £ 3.3% for M2-S341A and HSV-WT, and £ 2.1% for M2-R347D and HSV-WT). Login to comment