ABCMdb

ABCC7 p.Pro750Ala

None has been submitted yet.

Publications

PMID: 10692317 [PubMed] Xie J et al: "Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings."

No. Sentence Comment

33 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
57 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
147 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 Mutants P740A and P759A CFTR have similar open probability to WT CFTR, whereas P750A CFTR has significantly increased Po. Login to comment
162 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 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 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 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
196 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 (B) Representative single channel currents through a double proline mutant (P2A: P750A/P759A) are plotted. Login to comment
226 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 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
237 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 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
34 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
58 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
148 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
150 Mutants P740A and P759A CFTR have similar open probability to WT CFTR, whereas P750A CFTR has significantly increased Po. Login to comment
166 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
198 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
206 (B) Representative single channel currents through a double proline mutant (P2A: P750A/P759A) are plotted. Login to comment
228 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
231 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
241 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