ABCMdb

PMID: 17293558

St Aubin CN, Zhou JJ, Linsdell P
Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Mol Pharmacol. 2007 May;71(5):1360-8. Epub 2007 Feb 9., [PubMed]

Sentences

No. Mutations Sentence Comment

92 ABCC7 p.Lys95Gln Thus, the point mutation K95Q greatly weakened the blocking effects of glibenclamide, DNDS, lonid- amne, NPPB, and TLCS (Linsdell, 2005). Login to comment 95 ABCC7 p.Arg303Gln As shown in Fig. 5, a mutation that removed one of these positive charges, R303Q, significantly weakened the inhibitory effects of suramin. Login to comment 97 ABCC7 p.Arg303Glu The greatest weakening of inhibition was observed in the side chain charge-reversing R303E mutant, whereas the Fig. 4. Login to comment 105 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln A, example of leak-subtracted current-voltage relationships for K95Q and R303Q-CFTR recorded before (control) and after (ϩ suramin) the addition of 10 ␮M suramin to the intracellular solution. Login to comment 106 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln B, mean fraction of control current remaining (I/I0) after the addition of different concentrations of suramin at a membrane potential of -100 mV in K95Q (F) and R303Q (E). Login to comment 107 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Mean of data from five to six patches is shown, fitted by eq. 1 (solid lines), giving Kd values of 11.6 ␮M for K95Q and 48.2 ␮M for R303Q. Login to comment 109 ABCC7 p.Arg303Lys charge-conserving R303K mutant did not significantly affect the Kd value for suramin inhibition (Fig. 6). Login to comment 115 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Figure 7 compares the blocking effects of intracellular glibenclamide in wild-type CFTR with the charge-neutralizing mutants R303Q and K95Q. Login to comment 116 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln It can be seen that glibenclamide inhibition is weakened in R303Q, although the effects of this mutant are not as dramatic as those seen in K95Q. Login to comment 117 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Furthermore, a double mutant in which both of these positively charged residues were mutated to neutral glutamines (K95Q/R303Q) showed glibenclamide sensitivity similar to that of K95Q alone (Fig. 7). Login to comment 119 ABCC7 p.Arg303Gln As shown in Fig. 8, DNDS block was also weakened in R303Q; however, this mutation did not significantly affect block by lonidamine, NPPB, or TLCS. Login to comment 120 ABCC7 p.Arg303Gln The overall effects of the R303Q mutation on the affinity of block by the six different substances used in the present study are summarized in Fig. 9 and Table 1; the structures of these six substances are shown in Fig. 1. Login to comment 128 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln ABCC7 p.Arg303Gln A, example of leak-subtracted current-voltage relationships for wild-type, R303Q, K95Q, and the K95Q/R303Q double mutant recorded before (control) and after (ϩ glibenclamide) the addition of 30 ␮M glibenclamide to the intracellular solution. Login to comment 142 ABCC7 p.Arg303Gln Inhibition of R303Q-CFTR by open-channel blockers. Login to comment 143 ABCC7 p.Arg303Gln A, example of leak-subtracted current-voltage relationships for wild type and R303Q-CFTR recorded before (control) and after the addition of the named blocker to the intracellular solution: DNDS (100 ␮M), lonidamine (100 ␮M), NPPB (50 ␮M), or TLCS (50 ␮M). Login to comment 144 ABCC7 p.Arg303Gln B, mean fraction of control current remaining (I/I0) after the addition of these concentrations of blockers in wild type (E) and R303Q (F). Login to comment 159 ABCC7 p.Arg303Gln Mean Kd values estimated at -100 mV for each of the six CFTR inhibitors used in the present study under identical ionic conditions for wild type (Ⅺ) and R303Q (f). Login to comment 163 ABCC7 p.Lys95Gln Data for block of K95Q by DNDS, lonidamine, NPPB, and TLCS were taken from Linsdell (2005). Login to comment 164 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Blocker K95Q R303Q Suramin 0 ϩϩ Glibenclamide ϩϩ ϩ DNDS ϩϩ ϩ Lonidamine ϩϩ 0 NPPB ϩϩ 0 TLCS ϩϩ 0 0, Ͻ2-fold change in Kd; ϩ, 2to 8-fold increase in Kd; ϩϩ, Ͼ8-fold increase in Kd. Login to comment 176 ABCC7 p.Arg303Gln The charge-neutralizing R303Q mutation greatly weakens suramin block, significantly weakens the blocking effects of glibenclamide and DNDS (but to a lesser extent than for suramin), and has no apparent effect on block by lonidamine, NPPB, or TLCS (Fig. 9 and Table 1). Login to comment 178 ABCC7 p.Arg303Gln The reasons for the minor effect of the R303Q mutation on inhibition by glibenclamide and DNDS are less clear. Login to comment 181 ABCC7 p.Arg303Gln Removal of this surface charge in R303Q would then reduce the rate of glibenclamide and DNDS entry into the pore and to their primary binding site near Lys95. Login to comment 190 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Whatever the mechanism, the far greater effects of the K95Q mutation on block by glibenclamide and DNDS relative to R303Q (Table 1) are consistent with the most important interaction underlying open-channel block by these two molecules being with Lys95. Login to comment