ABCMdb

PMID: 10692317

Xie J, Zhao J, Davis PB, Ma J
Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings.
Biophys J. 2000 Mar;78(3):1293-305., [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCC7 p.Pro759Ala ABCC7 p.Thr757Ser ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Site-specific mutations were constructed following the manufacturer`s instructions using the following three mutagenesis oligonucleotides (showed 5Ј to 3Ј, with mutated base underlined): P740A, G CTC AGA ATC TGC TAC TAA GGA CAG C (RsaI enzyme restriction site is destroyed); P750A, G GCT GAT GCG AGC CAG TAT CGC CTC (BsrI site is created); P759A/T757S, C CTG AAG CGT GGC CGG AGA GCT GAT (BsaHI site is created and BsrI site is destroyed). Login to comment 34 ABCC7 p.Pro759Ala ABCC7 p.Thr757Ser ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Site-specific mutations were constructed following the manufacturer`s instructions using the following three mutagenesis oligonucleotides (showed 5b18; to 3b18;, with mutated base underlined): P740A, G CTC AGA ATC TGC TAC TAA GGA CAG C (RsaI enzyme restriction site is destroyed); P750A, G GCT GAT GCG AGC CAG TAT CGC CTC (BsrI site is created); P759A/T757S, C CTG AAG CGT GGC CGG AGA GCT GAT (BsaHI site is created and BsrI site is destroyed). Login to comment 35 ABCC7 p.Thr757Ser Although mutation was intended to affect only proline residues, upon sequencing, an additional conservative mutation, T757S, was noted. Login to comment 36 ABCC7 p.Thr757Ser ABCC7 p.Thr757Ser Subsequently, the individual prolines were mutated in sequence, with and without the T757S mutation, and their function was examined. Login to comment 37 ABCC7 p.Thr757Ser ABCC7 p.Thr757Ser No difference in channel activity was produced for any mutant by the T757S substitution. Login to comment 38 ABCC7 p.Pro759Ala ABCC7 p.Thr757Ser ABCC7 p.Thr757Ser Data presented in this paper for mutants containing P759A mutation are for constructs that also contain the unexpected T757S mutation. Login to comment 39 ABCC7 p.Pro759Ala ABCC7 p.Thr757Ser Data presented in this paper for mutants containing P759A mutation are for constructs that also contain the unexpected T757S mutation. Login to comment 57 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Vesicle preparation Six 75 cm2 flasks of 293 HEK cells transfected with either pCEP4(WT) or CFTR mutants (P3A, P2A, P740A, P750A, P759A) vectors were harvested and lysed following the procedure described previously (Xie et al., 1995, 1996). Login to comment 58 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Vesicle preparation Six 75 cm2 flasks of 293 HEK cells transfected with either pCEP4(WT) or CFTR mutants (P3A, P2A, P740A, P750A, P759A) vectors were harvested and lysed following the procedure described previously (Xie et al., 1995, 1996). Login to comment 64 ABCC7 p.Pro759Ala ABCC7 p.Pro740Ala In experiments with WT, P3A, P2A, P740A, P750, and P759A, cis solution also contains 50 units/ml cAMP-dependent protein kinase A catalytic subunit. Login to comment 65 ABCC7 p.Pro759Ala ABCC7 p.Pro740Ala In experiments with WT, P3A, P2A, P740A, P750, and P759A, cis solution also contains 50 units/ml cAMP-dependent protein kinase A catalytic subunit. Login to comment 147 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala All three proline mutations are involved in the enhanced activity of the P3A CFTR channel To investigate which proline is critical for the increased open probability of the P3A channel, we constructed all three single proline mutants (P740A, P750A, P759A) and incorporated each of them into the planar lipid bilayer. Login to comment 148 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala All three proline mutations are involved in the enhanced activity of the P3A CFTR channel To investigate which proline is critical for the increased open probability of the P3A channel, we constructed all three single proline mutants (P740A, P750A, P759A) and incorporated each of them into the planar lipid bilayer. Login to comment 149 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Mutants P740A and P759A CFTR have similar open probability to WT CFTR, whereas P750A CFTR has significantly increased Po. Login to comment 150 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Mutants P740A and P759A CFTR have similar open probability to WT CFTR, whereas P750A CFTR has significantly increased Po. Login to comment 153 ABCC7 p.Pro740Ala P740A CFTR had Po similar to the wild type, while its mean open life time is significantly (p Ͻ 0.05) lower (closing faster) than that of the WT CFTR, so FIGURE 4 ATP-and PKA-dependent gating of the P3A CFTR channel. Login to comment 154 ABCC7 p.Pro740Ala P740A CFTR had Po similar to the wild type, while its mean open life time is significantly (p b0d; 0.05) lower (closing faster) than that of the WT CFTR, so FIGURE 4 ATPand PKA-dependent gating of the P3A CFTR channel. Login to comment 160 ABCC7 p.Pro740Ala the P740A mutant must have a higher opening rate. Login to comment 161 ABCC7 p.Pro740Ala the P740A mutant must have a higher opening rate. Login to comment 162 ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala The mean open lifetime of P750A is not different from that of WT CFTR, but open probability of P750A is significantly higher than wild-type CFTR, though still significantly (p ϭ 0.044) lower than P3A. Login to comment 163 ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala Thus, P750A alone does not entirely account for the P3A channel activity, and the other two prolines also contribute to the high Po of P3A CFTR. Login to comment 164 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala P759A CFTR shows no increase in open probability compared to the wild type, but its mean open life time is significantly longer than that of the wild-type CFTR. Login to comment 165 ABCC7 p.Pro759Ala ABCC7 p.Pro759Ala ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala To test whether mutation of prolines 750 and 759, but not proline 740, would have even higher Po than P3A by combining the increased channel opening of P750A with the increased open life time of P759A, the double proline mutant P2A CFTR (P750A/P759A) was made. Login to comment 166 ABCC7 p.Pro759Ala ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala To test whether mutation of prolines 750 and 759, but not proline 740, would have even higher Po than P3A by combining the increased channel opening of P750A with the increased open life time of P759A, the double proline mutant P2A CFTR (P750A/P759A) was made. Login to comment 167 ABCC7 p.Pro759Ala P2A has an increased mean open life time, like the P759A mutant, but its Po is not significant higher than wild-type CFTR (p ϭ 0.46). Login to comment 168 ABCC7 p.Pro759Ala P2A has an increased mean open life time, like the P759A mutant, but its Po is not significant higher than wild-type CFTR (p afd; 0.46). Login to comment 196 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala The double (P2A; Fig. 10, lane 4) or single proline mutants (P740A, P750A, P759A; Fig. 10, lanes 5-7, respectively) were also fully glycosylated to a similar extent. Login to comment 198 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala The double (P2A; Fig. 10, lane 4) or single proline mutants (P740A, P750A, P759A; Fig. 10, lanes 5-7, respectively) were also fully glycosylated to a similar extent. Login to comment 204 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala (B) Representative single channel currents through a double proline mutant (P2A: P750A/P759A) are plotted. Login to comment 206 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala (B) Representative single channel currents through a double proline mutant (P2A: P750A/P759A) are plotted. Login to comment 226 ABCC7 p.Pro750Ala The properties of the P750A mutant are most similar to cyclophilin A-treated WT CFTR FIGURE 8 Open probability and mean open life time of WT and CFTR proline mutants. Login to comment 228 ABCC7 p.Pro750Ala The properties of the P750A mutant are most similar to cyclophilin A-treated WT CFTR FIGURE 8 Open probability and mean open life time of WT and CFTR proline mutants. Login to comment 229 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala The averaged values were Po ϭ 0.204 Ϯ 0.036 (WT), 0.438 Ϯ 0.029 (WT ϩ CyP (Cyclophilin A)), 0.577 Ϯ 0.090 (P3A CFTR), 0.161 Ϯ 0.018 (P740A), 0.426 Ϯ 0.030 (P750A), 0.166 Ϯ 0.034 (P759A), 0.272 Ϯ 0.059 (P2A), respectively. Login to comment 231 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala The averaged values were Po afd; 0.204 afe; 0.036 (WT), 0.438 afe; 0.029 (WT af9; CyP (Cyclophilin A)), 0.577 afe; 0.090 (P3A CFTR), 0.161 afe; 0.018 (P740A), 0.426 afe; 0.030 (P750A), 0.166 afe; 0.034 (P759A), 0.272 afe; 0.059 (P2A), respectively. Login to comment 237 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala It is also FIGURE 10 Heterologous expression of wild-type CFTR and proline mutants in 293 HEK cells: 293 HEK cells transfected with pCEP4(WT), pCEP4(P3A), pCEP4(P2A), pCEP4(P740A), pCEP4(P750A), or pCEP4(P759A) were immunoprecipitated and blotted as described in the Materials and Methods; mAb24-1 that recognizes the C-terminus of CFTR was used in the immunoprecipitation/Western blot. Login to comment 239 ABCC7 p.Pro759Ala ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala ABCC7 p.Pro740Ala Lane 1 (UNT): untransfected 293 HEK cells; lane 2: WT CFTR expressing cells; lanes 3-7: P3A, P2A, P740A, P750A, and P759A CFTR expressing cells, respectively. Login to comment 241 ABCC7 p.Pro759Ala ABCC7 p.Pro750Ala ABCC7 p.Pro740Ala Lane 1 (UNT): untransfected 293 HEK cells; lane 2: WT CFTR expressing cells; lanes 3-7: P3A, P2A, P740A, P750A, and P759A CFTR expressing cells, respectively. Login to comment 272 ABCC7 p.Pro574His ABCC7 p.His949Tyr ABCC7 p.Asp836* Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment 274 ABCC7 p.Pro574His ABCC7 p.His949Tyr ABCC7 p.Asp836* Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment