ABCMdb

PMID: 17582383

Melin P, Hosy E, Vivaudou M, Becq F
CFTR inhibition by glibenclamide requires a positive charge in cytoplasmic loop three.
Biochim Biophys Acta. 2007 Oct;1768(10):2438-46. Epub 2007 May 21., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Lys978Ser ABCC7 p.Lys978Gln ABCC7 p.Lys978Ala Charge-neutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTRinh-172. Login to comment 3 ABCC7 p.Lys978Arg The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Login to comment 4 ABCC7 p.Arg975Ala ABCC7 p.Arg975Ser ABCC7 p.Arg975Gln Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. Login to comment 77 ABCC7 p.Lys978Gln ABCC7 p.Lys978Arg ABCC7 p.Lys978Ala We investigated the role of the charged residue K978 in the sequence ILNRFSKD of CL3 (Fig. 1B) described as a region physically close to the pore [29] and examined the interaction of glibenclamide with mutated EGFP-CFTR channels: K978A, K978Q, K978R, Fig. 2. Login to comment 78 ABCC7 p.Lys978Ser Effect of K978S mutation on the whole cell EGFP-CFTR chloride currents. Login to comment 79 ABCC7 p.Lys978Ser Representative traces of global currents recorded on HEK-293 transfected with EGFP-CFTR channels wild-type (left) and charge neutralizing mutant K978S (right) are shown in presence of Fsk 10 bc;M (A), after perfusion of glibenclamide 100 bc;M (B), and sub-sequential addition of CFTRinh-172 10 bc;M (C). Login to comment 80 ABCC7 p.Lys978Ser (D) Corresponding current-voltage relationships normalized by cell capacitance (n=6 for wt, n=10 for K978S). Login to comment 81 ABCC7 p.Lys978Ser K978S compared to CFTR-wt. Login to comment 84 ABCC7 p.Lys978Ser ABCC7 p.Lys978Gln ABCC7 p.Lys978Arg ABCC7 p.Lys978Ala Similar effects in the presence of Fsk were recorded with cells expressing K978A, K978Q, K978R and K978S mutants CFTR. Login to comment 85 ABCC7 p.Lys978Ser Representative chloride currents recorded with K978S are shown in Fig. 2A (right). Login to comment 87 ABCC7 p.Lys978Ser Interestingly, perfusion of glibenclamide did not induce inhibition of forskolin-activated chloride currents in EGFP-CFTR-K978S expressing cells (Fig. 2B right). Login to comment 88 ABCC7 p.Lys978Ser The difference between the two I/V curves in the presence of Fsk versus Fsk+Glib, at all voltages for K978S channels, was not statistically different (PN0.05) using analysis of variance followed by a Bonferroni post hoc test (Fig. 2D right). Login to comment 92 ABCC7 p.Lys978Ser In contrast, K978S activity, after glibenclamide application, was fully inhibited by CFTRinh-172 (Fig. 3A, right). Login to comment 93 ABCC7 p.Lys978Ser From continuous whole cell recordings with voltage steps between -40 mV and +40 mV, we estimated that full inhibition of CFTR-wt current by glibenclamide was achieved in ~250 s (Fig. 3B left) whereas K978S activity remained remarkably stable as long as the sulfonylurea was present (Fig. 3B right). Login to comment 94 ABCC7 p.Lys978Ser The inhibition by CFTRinh-172 of CFTR-K978S was, on the contrary, almost immediate and very rapid as illustrated at the end of the recording, Fig. 3B (right). Login to comment 95 ABCC7 p.Lys978Ser Thus, the charge neutralizing mutant K978S is resistant to glibenclamide block, but remains sensitive to the blocker CFTRinh-172. Login to comment 96 ABCC7 p.Lys978Gln ABCC7 p.Lys978Arg ABCC7 p.Lys978Ala To investigate the role of the charge of the side chain of K978 in glibenclamide resistance, different amino acid substitutions were examined: K978A, K978Q, K978R. Login to comment 98 ABCC7 p.Lys978Ser ABCC7 p.Lys978Gln ABCC7 p.Lys978Arg ABCC7 p.Lys978Ala Fig. 4A presents the ratio Iglib/Ifsk, recorded at -100 mV, with 100 bc;M glibenclamide, for K978A, K978Q, K978R, K978S channels compared to CFTR-wt. Login to comment 100 ABCC7 p.Lys978Arg Indeed, the ratio at -100 mV for the charge conservative mutant K978R (0.17&#b1; 0.04, n=4) was not significantly different from that of CFTR-wt. Login to comment 101 ABCC7 p.Lys978Ser However, for K978S channels, the ratio at this voltage was significantly (Pb0.001) increased to 0.81&#b1;0.04 (n=4). Login to comment 104 ABCC7 p.Lys978Ser (A) Summary of mean current densities (pA/pF), recorded at -100 mV and +40 mV, in various conditions (indicated on graph) for HEK cells transfected with EGFP-CFTR-wt (left) or EGFP-CFTR-K978S (right). Login to comment 105 ABCC7 p.Lys978Ser Data are mean&#b1;S.E.M. of n experiments (n=7 for wt, n=10 for K978S). Login to comment 107 ABCC7 p.Lys978Ser Note that EGFP-CFTR-K978S channels were inhibited by CFTRinh-172 but not by glibenclamide. Login to comment 108 ABCC7 p.Lys978Gln ABCC7 p.Lys978Ala neutralizing mutations (K978A, K978Q) rendered channels insensitive to inhibition by glibenclamide as demonstrated by the time course of current recorded at +40 mV (Fig. 4B). Login to comment 114 ABCC7 p.Arg975Ala ABCC7 p.Arg975Ser ABCC7 p.Arg975Gln After expression in HEK-293 cells, the whole-cell chloride currents recorded with charge neutralizing mutants CFTR-R975A, R975Q and R975S (Fig. 5) showed similar electrophysiological properties as CFTR-wt: linear activation with Fsk, inhibition by glibenclamide and by CFTRinh-172. Login to comment 119 ABCC7 p.Lys978Ser For K978S channels, refractory to glibenclamide, the experimental reversal potentials were -43&#b1;1.5 mV (n=4) for bromide, -42.5&#b1; 1.5 mV (n=4) for chloride, and -29&#b1;5 mV (n=3) for iodide. Login to comment 128 ABCC7 p.Lys978Gln ABCC7 p.Lys978Ala (B) Examples of time course of current densities (between +40 mVand -40 mV) from the charge neutralizing mutants CFTR-K978Q (a0;) and CFTR-K978A (ef;) in presence of Fsk 10 bc;M, and Fsk+Glib 100 bc;M, and Fsk+CFTRinh-172 10 bc;M. Login to comment 138 ABCC7 p.Lys978Ser ABCC7 p.Lys978Gln ABCC7 p.Lys978Ala This was the case for the mutations that neutralized the charge of the side chain of the residue 978 (K978A, K978Q, K978S). Login to comment 140 ABCC7 p.Lys978Arg In contrast, substitution of lysine 978 with another positively charged residue (K978R) had no incidence on glibenclamide sensitivity of CFTR channels. This charge conservative mutant shared the same properties of inhibition as CFTRwt. Login to comment 151 ABCC7 p.Arg975Ala Original currents recorded on HEK-293 transfected with EGFP-CFTR-R975A with Fsk 10 bc; M (A), Fsk+Glib 100 bc;M (B), and Fsk+CFTRinh-172 10 bc;M (C). Login to comment 152 ABCC7 p.Arg975Ala (D) Representative time course of current densities from the charge neutralizing mutant EGFP-CFTR-R975A in presence of Fsk 10 bc;M, Fsk+Glib 100 bc;M, Fsk+CFTRinh-172 10 bc;M. Login to comment 153 ABCC7 p.Arg975Ala ABCC7 p.Arg975Ser ABCC7 p.Arg975Gln (E) Current-voltage relationships normalized by cell capacitance recorded from R975A (n=11), R975S (n=7) and R975Q (n=4) EGFP-CFTR channels in presence of agonists. Login to comment