ABCMdb

PMID: 11160632

Al-Nakkash L, Hu S, Li M, Hwang TC
A common mechanism for cystic fibrosis transmembrane conductance regulator protein activation by genistein and benzimidazolone analogs.
J Pharmacol Exp Ther. 2001 Feb;296(2):464-72., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Lys1250Ala We conclude that benzimidazolone analogs and genistein act through a common mechanism, based on the following evidence: 1) both act only on phosphorylated CFTR, 2) the maximal ⌬F508-CFTR current activated by benzimidazolone analogs is identical to that induced by genistein, 3) benzimidazolone analogs increase the open probability of the forskolin-dependent ⌬F508-CFTR channel activity through an increase of the channel open time and a decrease of the channel closed time (effects indistinct from those reported for genistein), and 4) the prolonged K1250A-CFTR channel open time (in the presence of 10 ␮M forskolin) is unaffected by benzimidazolone analogs or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at nucleotide binding domain 2 (NBD2). Login to comment 3 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala From our studies with the double mutant ⌬F508/K1250A-CFTR, we conclude that benzimidazolone analogs and genistein rectify the ⌬F508-CFTR prolonged closed time independent of their effects on channel open time, since these agonists enhance ⌬F508/K1250A-CFTR activity by shortening the channel closed time. Login to comment 34 ABCC7 p.Lys1250Ala Neither benzimidazolone analogs nor genistein potentiate cAMP-dependent K1250A-CFTR activity, a nucleotide binding domain 2 mutant with a prolonged open time due to diminished ATP hydrolysis activity. Login to comment 35 ABCC7 p.Lys1250Ala Although the ⌬F508-CFTR channel open time is increased by introducing the K1250A mutation into the ⌬F508 background, the prolonged closed time is unaffected, suggesting that the prolonged closed time associated with the ⌬F508 mutation is independent of channel open time. Login to comment 36 ABCC7 p.Lys1250Ala Since benzimidazolone analogs and genistein enhance ⌬F508/K1250A-CFTR activity by shortening the channel closed time, we conclude that their rectification of the ⌬F508-CFTR prolonged closed time is independent of their effects on the channel open time. Login to comment 38 ABCC7 p.Lys1250Ala Materials and Methods Cell Culture NIH3T3 mouse fibroblast cells stably expressing either ⌬F508-CFTR or K1250A-CFTR were prepared as described previously (Berger et al., 1991; Zeltwanger et al., 1999). Login to comment 39 ABCC7 p.Lys1250Ala The ⌬F508/K1250A-CFTR double mutation was generated as follows. Login to comment 40 ABCC7 p.Lys1250Ala Plasmids containing ⌬F508-CFTR (⌬F508pRBG4) or K1250A-CFTR (K1250ApRBG4) were generously provided by Dr. R. R. Kopito (Stanford University, Stanford, CA). Login to comment 44 ABCC7 p.Lys1250Ala Using Superfect reagent (Qiagen, Valencia, CA), the ⌬F508/K1250A-CFTR double mutant was transiently transfected into NIH3T3 mouse fibroblast cells, according to the manufacturer`s protocol. Login to comment 188 ABCC7 p.Lys1250Ala Effects of NS004 and NS1619 on K1250A-CFTR. Login to comment 189 ABCC7 p.Lys1250Ala To further corroborate our evidence that NS004 and NS1619 act to stabilize the channel open state, we examined the effect of these drugs upon the K1250A-CFTR mutant channel. Login to comment 191 ABCC7 p.Lys1250Ala Figure 7 shows a representative recording of K1250A-CFTR in a cell-attached patch. Login to comment 193 ABCC7 p.Lys1250Ala Fold increases in mean K1250A-CFTR current amplitude were 1.09 Ϯ 0.11 (n ϭ 3) and 1.15 Ϯ 0.18 (n ϭ 3), respectively. Login to comment 195 ABCC7 p.Lys1250Ala In the same patch forskolin alone can reactivate the K1250A-CFTR current, and subsequent addition of 20 ␮M genistein had minimal effect on the current (fold increase ϭ 1.03 Ϯ 0.02, n ϭ 5). Login to comment 197 ABCC7 p.Lys1250Ala Thus, neither benzimidazolone analogs nor genistein altered the Po of K1250A-CFTR in the presence of a maximal concentration of forskolin. Login to comment 198 ABCC7 p.Lys1250Ala We further examined the effect of benzimidazolone analogs and genistein upon the open time of K1250A-CFTR. Login to comment 199 ABCC7 p.Lys1250Ala In cell-attached patches, steady-state macroscopic K1250A-CFTR current was generated by forskolin alone or forskolin plus either genistein or NS004 (each 20 ␮M); the patch was then excised into an ATP-free bath. Login to comment 203 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala These data demonstrate that the open time of the K1250A-CFTR channels activated with a maximally effective concentration of forskolin (10 ␮M) is not affected by either genistein or benzimidazolone analogs. Since neither the Po nor the open time of K1250A-CFTR is affected by genistein or benzimidazolone analogs, we conclude that the closed time of K1250A-CFTR is not affected when the cAMP pathway is maximally activated. Login to comment 204 ABCC7 p.Lys1250Ala The fact that these drugs do not change the open time of K1250A-CFTR is consistent with the idea that genistein acts by inhibiting ATP hydrolysis at NBD2 (Wang et al., 1998; Randak et al., 1999). Login to comment 205 ABCC7 p.Lys1250Ala However, a lack of effect on the K1250A-CFTR mutant could be caused by an obliteration of the binding site for these compounds by the mutation. Login to comment 206 ABCC7 p.Lys1250Ala This is perhaps not the case, since these reagents increase K1250A-CFTR channel activity when the cAMP stimulation is submaximal. Login to comment 207 ABCC7 p.Lys1250Ala Like wt-CFTR, the activity of K1250A-CFTR can be manipulated using different concentrations of forskolin (Al-Nakkash and Hwang, 1999). Login to comment 208 ABCC7 p.Lys1250Ala For example, sequential addition of 100 nM and then 10 ␮M forskolin to the bath solution generates an incremental increase in the steady-state macroscopic K1250A-CFTR current (data not shown). Login to comment 209 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Under conditions that produce submaximal stimulation of the cAMP-dependent K1250A-CFTR channel activation (i.e., 100 nM forskolin), benzimidazolone analogs enhanced mean K1250A-CFTR current. Login to comment 210 ABCC7 p.Lys1250Ala In the presence of 100 nM forskolin, 50 nM and 20 ␮M NS004 increased the K1250A-CFTR current by 6.22 Ϯ 2.55-fold (n ϭ 6) and 19.09 Ϯ 9.71-fold (n ϭ 6), respectively. Login to comment 212 ABCC7 p.Lys1250Ala These data suggest, for those K1250A-CFTR channels that are not maximally stimulated (i.e., have not attained a maximum Po), that benzimidazolone analogs can potentiate the cAMP-dependent channel current. Login to comment 213 ABCC7 p.Lys1250Ala Effect of NS004 and NS1619 upon the ⌬F508/K1250A Double Mutation. Login to comment 217 ABCC7 p.Lys1250Ala To address this, we examined the effects of benzimidazolone analogs and genistein on the double mutant ⌬F508/K1250A-CFTR. Login to comment 218 ABCC7 p.Lys1250Ala Figure 8A shows a 30-min cell-attached recording from an NIH3T3 cell expressing ⌬F508/K1250A-CFTR. Login to comment 224 ABCC7 p.Lys1250Ala Effect of NS1619 and genistein upon forskolin-dependent K1250A-CFTR channel current. Login to comment 225 ABCC7 p.Lys1250Ala In this continuous cell-attached recording (lasting 50 min), the application of 10 ␮M forskolin elicits a macroscopic K1250A-CFTR current. Login to comment 227 ABCC7 p.Lys1250Ala Similarly, there is no effect of 20 ␮M genistein upon the steady-state macroscopic K1250A-CFTR current generated by 10 ␮M forskolin. Login to comment 228 ABCC7 p.Lys1250Ala The expanded section shows the closure of all K1250A channels upon removal of agonists and a return to basal activity (expanded trace lasts for 200 s). Login to comment 230 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala K1250A mutation into the ⌬F508-CFTR background does not rectify the functional defect associated with ⌬F508-CFTR and that NS004 greatly potentiates the Po of ⌬F508/K1250A-CFTR. Login to comment 232 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala In this cell-attached patch recording of ⌬F508/K1250A-CFTR, one single channel opens for 20 s in the presence of forskolin alone (a phenotype for the K1250A-CFTR mutation), but the channel is predominantly closed (a phenotype for the ⌬F508-CFTR mutation). Login to comment 263 ABCC7 p.Lys1250Ala Effect of NS004 and NS1619 on ⌬F508/K1250A current. Login to comment 264 ABCC7 p.Lys1250Ala A, 30-min continuous recording showing the effect of NS004 on forskolin-dependent macroscopic ⌬F508/K1250A-CFTR current. Login to comment 267 ABCC7 p.Lys1250Ala B, effect of NS1619 on forskolin-dependent ⌬F508/ K1250A-CFTR channel current in a patch containing one single channel. Login to comment 273 ABCC7 p.Lys1250Ala Last, neither benzimidazolone analogs nor genistein can potentiate K1250A-CFTR channel current activated by a maximally effective concentration of forskolin. Login to comment 275 ABCC7 p.Lys1250Ala We show, from macroscopic relaxation analysis, that the open time for K1250A-CFTR is not changed by benzimidazolone analogs or genistein, supporting the hypothesis that these compounds stabilize the open state by inhibiting ATP hydrolysis at NBD2. Login to comment 285 ABCC7 p.Lys1250Ala First, when K1250A-CFTR is submaximally stimulated with nanomolar forskolin, the closed time can still be decreased by benzimidazolone analogs. Login to comment 286 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Second, when the double mutant ⌬F508/ K1250A-CFTR is stimulated with a maximally effective concentration of forskolin, the prolonged open time caused by the K1250A mutation does not automatically correct the abnormally long closed time associated with the ⌬F508 mutation. Login to comment