ABCMdb

PMID: 17381427

Pereira MM, Parker J, Stratford FL, McPherson M, Dormer RL
Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP.
Biochem J. 2007 Jul 1;405(1):181-9., 2007-07-01 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Thr421Ala Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 µM. Login to comment 39 ABCC7 p.Thr421Ala The T421A mutation was introduced into the pRSETB vector using Stratagene`s QuikChange® II Site-directed Mutagenesis kit and oligonucleotide primers (forward and reverse) containing the mutation amidst a 21 base-pair 5 - and 3 - flanking region. Login to comment 154 ABCC7 p.Thr421Ala However, as for ATP binding, cAMP displacement followed a biexponential curve, yielding values for half-maximal displacement of [3 H]cAMP of 2.6 and 167 µM. Sullivan et al. [18] compared the whole-cell chloride channel activity elicited by injection of 50 µM cAMP into cells expressing wild-type CFTR or the mutant form, T421A. Login to comment 157 ABCC7 p.Thr421Ala We introduced the T421A mutation into CFTR cDNA (see the Experimental section) and expressed an NBD1-R domain protein to investigate the effect on cAMP binding. Login to comment 158 ABCC7 p.Thr421Ala As shown in Figure 5(B), the T421A mutant protein bound [3 H]cAMP but showed half-maximal displacement by unlabelled cAMP at 9.2 and 201 µM. Login to comment 159 ABCC7 p.Thr421Ala Thus the results suggest that the T421A mutation results in decreased affinity of the higher affinity binding, which is responsible for activation of CFTR. Login to comment 177 ABCC7 p.Thr421Ala All the results are means + - S.E.M.; (A) n = 7 for displacement by cAMP; (B) displacement by cAMP from wild-type (closed circles, n = 5) or T421A (open squares, n = 5) NBD1-R domain protein. Login to comment 218 ABCC7 p.Thr421Ala ABCC7 p.Thr421Ala We synthesized a T421A mutant form of the NBD1-R domain protein and showed a decreased affinity for cAMP binding (Figure 5B), consistent with the demonstration that the full-length T421A CFTR showed reduced chloride channel activity in whole cells [18]. Login to comment