ABCMdb

PMID: 11889571

Gupta J, Linsdell P
Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.
Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Login to comment 5 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. Login to comment 63 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala While block of the TM12 mutants S1141A (Fig. 1) and T1134A and M1137A (data not shown) was indistinguishable from wild-type, block was significantly weakened in the TM6 mutants F337A and T338A, and significantly strengthened in the TM12 mutants N1138A and T1142A (Fig. 1). Login to comment 69 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Mean fraction of control current remaining following addition of 60 µM glibenclamide (I/I0) is shown as a function of voltage for wild-type (q), T338A (s), N1138A (s), F337A (ss) and T1142A (xx). Login to comment 70 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Mean of data from 5-10 patches, fitted by Eq. according to the mean parameters shown in Fig. 3 rent remaining following addition of glibenclamide (I/I0) was significantly reduced at all voltages in N1138A and T1142A (P<0.05), and significantly increased in F337A and T338A at negative membrane potentials (P<0.05). Login to comment 71 ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala In contrast, I/I0 was not altered in T1134A, M1137A or S1141A at any voltage (data not shown). Login to comment 75 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Consistent with the results shown in Fig. 2, Kd(0) was significantly increased in F337A and T338A, and significantly decreased in N1138A and T1142A (Fig. 3A). Login to comment 83 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala The extracellular Cl-concentration had a similar effect on Kd(0) in the TM6 mutants F337A (Fig. 5A) and T338A (Figs. 4, 5A). Login to comment 84 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala In contrast, reducing extracellular Cl-concentration from 154 mM to 10 mM had no significant effect on apparent glibenclamide affinity in the TM12 mutants N1138A (Fig. 5A) or T1142A (Figs. 4, 5A). Login to comment 85 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala As a result, the effect of these two mutations on Kd(0) was dependent on the extracellular Cl-concentration: in N1138A, Kd(0) was reduced to 35% of the wild-type value with 154 mM Cl- but only to 61% of wild-type with 10 mM Cl-, while in T1142A, Kd(0) was 40% of wild-type with 154 mM Cl- and 50% with 10 mM Cl-. Login to comment 98 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr1142Ala ABCC7 p.Thr1142Ala A Example I-V relationships for wild-type (left), T338A (center) and T1142A (right), recorded with 10 mM extracellular Clas described in Materials and methods, before (Ctrl) and immediately following addition of 60 µM glibenclamide (+Glib) to the intracellular solution. B Mean fraction of control current remaining following addition of glibenclamide, with 154 mM (q) or 10 mM (qq) extracellular Cl-, for wild-type (left), T338A (center) and T1142A (right). Login to comment 100 ABCC7 p.Asn1138Ala A similar lack of extracellular Cl-dependence was observed in N1138A (data not shown) Discussion Glibenclamide causes a relatively high affinity block of CFTR [32, 34] and other Cl-channels [14, 29, 31, 42]. Login to comment 106 ABCC7 p.Phe337Ala ABCC8 p.Thr338Ala Two mutations in TM6, F337A and T338A, significantly weakened block by glibenclamide (Figs. 2, 3); this effect was apparently independent of the extracellu- 744 Fig. 5 Extracellular Cl-dependence of the apparent affinity and voltage dependence of glibenclamide block. Login to comment 112 ABCC7 p.Phe337Ala However, the increase in Kd(0) was modest in both cases (1.5-fold increase for F337A compared to wild-type, and 2.1-fold increase for T338A), and both mutants are still clearly blocked in a voltage-dependent manner by glibenclamide (Figs. 1, 2). Login to comment 115 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala In contrast to the effects of these mutations in TM6, two mutations in TM12 (N1138A and T1142A) caused a significant increase in the apparent affinity of glibenclamide block (Figs. 2, 3). Login to comment 116 ABCC7 p.Thr1134Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Met1137Ala This does not appear to be a nonspecific effect of mutagenesis within TM12, since three other mutations in this region (T1134A, M1137A, S1141A) had no effect on glibenclamide block (Fig. 3). Login to comment 120 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Two kinds of interactions may underlie the ability of extracellular Cl- ions to weaken glibenclamide block of wild-type, but not N1138A or T1142A CFTR. Login to comment 122 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala In this case, N1138A and T1142A might alter the structure of a binding site for both Cl- and glibenclamide, reducing its affinity for Cl- ions and thereby indirectly increasing glibenclamide occupancy of the pore. Login to comment 132 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala However, it is interesting to note that both suggest that: (1) the mutations N1138A and T1142A can increase the apparent affinity of glibenclamide block without directly affecting the interaction between glibenclamide and the pore walls, and (2) these two mutations both interfere with Cl-binding within the pore. Login to comment 134 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala Furthermore, the suggestion that TM12 residues N1138 and T1142 contribute to Cl-binding within the pore conflicts with our previous finding that the mutations N1138A and T1142A have no effect on unitary Cl-conductance [10]. Login to comment 135 ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala Interestingly, these two mutations did not affect SCN- block, although other TM12 mutants (T1134A, M1137A, S1141A) did [10], suggesting that Cl- and SCN- binding may be controlled by different structural features within TM12. Login to comment 147 ABCC7 p.Arg347Asp Am J Physiol 271:C628-C634 20. Linsdell P, Hanrahan JW (1997) Interaction of channel blockers with R347D-CFTR [abstract]. Login to comment