ABCMdb

PMID: 10880569

Ikuma M, Welsh MJ
Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8675-80., 2000-07-18 [PubMed]

Sentences

No. Mutations Sentence Comment

36 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala In CFTR, the NBD1 mutation K464A reduces ATPase activity to Ϸ15%, and the NBD2 mutation K1250A eliminates ATPase activity (24). Login to comment 39 ABCC7 p.Lys1250Ala Strikingly, a variant with both NBDs mutated (K464A͞K1250A) showed significant activity and gating not different from CFTR-K1250A (10). Login to comment 123 ABCC7 p.Lys1250Ala Prolongation of the burst duration is similar to the result of two other interventions that allow nucleotide binding but not hydrolysis: binding of the nonhydrolyzable AMP-PNP (9, 10, 16, 17, 38) and an NBD2 mutation that prevents hydrolysis, K1250A (10, 11, 14, 24). Login to comment 127 ABCC7 p.Asp1370Asn We studied the CFTR-D1370N mutant; this channel has reduced activity (11, 39). Login to comment 128 ABCC7 p.Asp572Asn We did not study the analogous mutation in NBD1, D572N, because it shows defective biosynthesis and is not processed to the cell surface. Login to comment 129 ABCC7 p.Asp1370Asn We predicted that CFTR-D1370N would not discriminate between ATP, MgATP, and CaATP. Login to comment 136 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala We tested variants with mutations in the Walker A lysine, CFTR-K464A and -K1250A. Login to comment 140 ABCC7 p.Lys1250Ala With MgATP, CFTR-K1250A showed prolonged bursts (Fig. 3 B and C), as previously reported (10, 11, 14, 24). Login to comment 143 ABCC7 p.Lys464Ala In contrast, with CFTR-K464A, the burst duration was the same with MgATP and ATP alone (Fig. 3 B and C). Login to comment 144 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala There are two potential explanations for the difference between K464A and K1250A. Login to comment 146 ABCC7 p.Lys464Ala Alternatively, in K464A, most of the gating may be due to ATP interactions with NBD2. Login to comment 148 ABCC7 p.Asp1370Asn Effect of MgATP, ATP alone, and CaATP on Cl- current of CFTR-D1370N. Login to comment 155 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Therefore, we studied CFTR-K1250A and CFTR-K464A at two different ATP concentrations. Login to comment 156 ABCC7 p.Lys1250Ala With 1 mM ATP and 4 mM Mg2ϩ , CFTR-K1250A showed prolonged bursts, but with a low MgATP concentration (5-20 ␮M ATP and 4 mM Mg2ϩ ), burst duration decreased (Fig. 4 A and C). Login to comment 159 ABCC7 p.Lys1250Ala This might prevent the channel from entering the prolonged bursts that result from NBD2 gating in K1250A. Login to comment 161 ABCC7 p.Lys464Ala Yet, prolonged burst durations have not been observed with K464A (10, 11, 14, 24). Login to comment 164 ABCC7 p.Lys464Ala However, if NBD1 has a higher ATP affinity than NBD2, this premise predicts that K464A would have long bursts at low MgATP concentrations. Login to comment 166 ABCC7 p.Lys464Ala With 1 mM ATP and 4 mM Mg2ϩ , CFTR-K464A showed durations approximately the same as observed with wild-type CFTR. Login to comment 189 ABCC7 p.Lys1250Ala The K1250A variant has a similar effect, blocking the O1 3 O2 transition and thereby prolonging bursts with MgATP (Fig. 3). Login to comment 190 ABCC7 p.Lys1250Ala In contrast, with ATP alone, the O1 state of K1250A is unstable and ATP dissociates more quickly (Fig. 3). Login to comment 192 ABCC7 p.Lys1250Ala This result suggests that the K1250A mutation reduces ATP binding to NBD2. Login to comment 194 ABCC7 p.Asp1370Asn It is also noteworthy that MgATP did not stimulate current to a greater extent than ATP alone when CFTR had a mutation in NBD2 (D1370N) that disrupts divalent cation binding (Fig. 2). Login to comment 198 ABCC7 p.Lys464Ala Our finding that the K464A mutation can generate bursts with a prolonged duration (Fig. 4) supports this conclusion. Login to comment 203 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Effect of MgATP and ATP alone on CFTR-K1250A and -K464A. Login to comment 238 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Effect of ATP concentration on gating of CFTR-K1250A (A and C) and K464A (B and D) channels. Login to comment