ABCMdb

PMID: 15634668

Linsdell P
Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5., 2005-03-11 [PubMed]

Sentences

No. Mutations Sentence Comment

69 ABCC7 p.Lys95Arg ABCC7 p.Lys95Glu In a striking parallel with previous findings involving Arg-334 (13, 16), mutagenesis of Lys-95 had a strongly charge-dependent effect on I-V rectification (Fig. 1); the charge-conservative K95R, like wild type CFTR, showed a practically linear I-V relationship, whereas all of the other mutations, and especially the charge-reversing K95E, caused significant outward rectification. Login to comment 74 ABCC7 p.Lys95Gln A, example leak-subtracted current-voltage relationships for wild type CFTR and K95Q-CFTR following maximal channel activation with the protein kinase A catalytic subunit and PPi. In both cases, currents were recorded before (control) and after (ϩ glibenclamide) the addition of 30 ␮M glibenclamide to the intracellular solution. Login to comment 75 ABCC7 p.Lys95Gln B, mean fraction of control current remaining (I/I0) after the addition of different concentrations of glibenclamide in wild type (E) and K95Q (●) at a membrane potential of -100 mV. Login to comment 76 ABCC7 p.Lys95Gln Values are means of data from 3-5 patches fitted as described under "Experimental Procedures," with a Kd of 12.6 ␮M and an nH of 1.07 for wild type and a Kd of 107 ␮M for K95Q. Login to comment 77 ABCC7 p.Lys95Gln Because the weak blocking effects of glibenclamide meant that a full concentration-inhibition curve could not be obtained for K95Q, nH was fixed at 1. Login to comment 78 ABCC7 p.Lys95Gln C, voltage dependence of glibenclamide block in wild type (E) and K95Q (●). Login to comment 83 ABCC7 p.Lys95Met Because of low current expression in baby hamster kidney cell membrane patches, K95M was not included in this analysis. Login to comment 85 ABCC7 p.Lys95Gln As shown in Fig. 2, inhibition of macroscopic CFTR current by glibenclamide, a potent intracellular open channel blocker, was greatly weakened in K95Q as compared with wild type. Login to comment 87 ABCC7 p.Lys95Gln In K95Q, the Kd at this voltage was increased to 107 ␮M, an increase of ϳ8.5-fold. Login to comment 88 ABCC7 p.Lys95Gln Furthermore, glibenclamide block of K95Q appeared only weakly dependent on voltage (Fig. 2C). Login to comment 91 ABCC7 p.Lys95Gln ABCC7 p.Lys95Arg ABCC7 p.Lys95Glu ABCC7 p.Lys95Cys ABCC7 p.Lys95Ala These results, using a number of different amino acid substitutions of Lys-95, strongly suggest that side chain charge at this position is important in controlling the apparent affinity of glibenclamide block; the apparent Kd at -100 mV was not affected in the charge-conservative K95R but was significantly increased in charge-neutralizing mutants (K95A, K95C, K95Q) and most strongly increased in the charge-reversing K95E mutant. Login to comment 95 ABCC7 p.Lys95Gln A, example leak-subtracted current-voltage relationships for wild type and K95Q-CFTR following maximal channel activation with the protein kinase A catalytic subunit and PPi. In each case, currents were recorded before (control) and after the addition of different CFTR channel blockers to the intracellular solution, namely 4,4Ј-dinitrostilbene-2,2Ј-disulfonic acid (DNDS) (100 ␮M), lonidamine (100 ␮M), NPPB (50 ␮M), or taurolithocholate-3-sulfate (TLCS) (50 ␮M). Login to comment 96 ABCC7 p.Lys95Gln B, mean fraction of control current remaining (I/I0) after the addition of these concentrations of blockers in wild type (E) and K95Q (●) as a function of membrane potential. Login to comment 98 ABCC7 p.Lys95Gln channel blockers on wild type and K95Q-CFTR are compared in Fig. 4. Login to comment 101 ABCC7 p.Lys95Gln In contrast, at the same concentrations each of these substances had only weak inhibitory effects on K95Q-CFTR. Login to comment 103 ABCC7 p.Lys95Gln Although this provides a rough approximation only, particularly as the estimated Kd was far greater than the concentration of blocker actually used in the case of K95Q, it does suggest that the blocking effects of each of these substances is in fact considerably more sensitive to mutagenesis of Lys-95 than are the blocking effects of glibenclamide. Login to comment 123 ABCC7 p.Lys95Gln In contrast, the very low sensitivity of K95Q to other open channel blockers suggests that Lys-95 plays a dominant role in coordinating binding of these substances within the pore. Login to comment