ABCMdb

PMID: 25267914

Csanady L, Torocsik B
Structure-activity analysis of a CFTR channel potentiator: Distinct molecular parts underlie dual gating effects.
J Gen Physiol. 2014 Oct;144(4):321-36. doi: 10.1085/jgp.201411246., [PubMed]

Sentences

No. Mutations Sentence Comment

32 ABCC7 p.Lys1250Ala Second, it increased by approximately threefold both the rate of opening of WT channels (step C1࢐O1) and the slow rate of nonhydrolytic closure (step O1࢐C1) of catalytically inactive mutants, such as K1250A CFTR in which lack of the conserved NBD2 Walker A lysine side chain abrogates ATP hydrolysis at site 2 (Ramjeesingh et al., 1999). Login to comment 46 ABCC7 p.Gly551Asp The first CFTR potentiator to enter clinical use, VX-770 (Ivacaftor; Vertex Pharmaceuticals [Van Goor et al., 2009]), was recently approved for the treatment of CF patients carrying the G551D (Ramsey et al., 2011) and other rare CFTR mutations. Login to comment 49 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser pore-block measurements on E1371S (see Fig. 2 A) or K1250A CFTR (see Figs. 3 and 4), surviving currents of channels opened in resting oocytes as the result of endogenous phosphorylation were also used (Csan&#e1;dy and T&#f6;r&#f6;csik, 2014). Login to comment 70 ABCC7 p.Lys1250Ala M A T E R I A L S A N D M E T H O D S Molecular biology WT and K1250A CFTR cDNA subcloned into the pGEMHE plasmid (Vergani et al., 2003) was linearized using NheI and transcribed in vitro using T7 polymerase (mMESSAGE kit; Ambion). Login to comment 77 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala MgATP (Sigma-Aldrich) was made up at 400 mM (adjusted to pH 7.1 with NMDG) and diluted into the bath solution at 2 and 10 mM final concentrations, respectively, for recordings on WT and K1250A CFTR (the higher [ATP] for K1250A was used to compensate for its reduced ATP-binding affinity [Vergani et al., 2003]). Login to comment 83 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser A convenient macroscopic assay for measuring fractional effects on average ion flux rates through bursting channels is provided by nonhydrolytic mutant CFTR channels such as E1371S (Vergani et al., 2003) or K1250A. Login to comment 86 ABCC7 p.Glu1371Ser As expected, application of MOPS&#e032; at a concentration of 80 mM, approximately ninefold its own KI (Fig. 2 A, green bars), reversibly reduced macroscopic current through locked-open E1371S channels by almost 90% (Fig. 2 A). Login to comment 87 ABCC7 p.Glu1371Ser In the presence of 80 mM MOPS&#e032; , addition of NPPB (Fig. 2 A, brown bars) further suppressed locked-open E1371S currents in a dose-dependent manner; however, further fractional reduction by 210 &#b5;M NPPB (&#e07a;10-fold its own KI) was only &#e07a;50% (Fig. 2 A, yellow box magnified in inset). Login to comment 106 ABCC7 p.Lys1250Ala We next probed acceleration by NPPB of the slow nonhydrolytic closing rate of K1250A CFTR channels (Fig. 2 F, gray bar) upon removal of ATP (Fig. 2 E, gray fit lines and time constants). Login to comment 110 ABCC7 p.Glu1371Ser (A) Macroscopic locked-open E1371S CFTR current at &#e032;120 mV after removal of ATP from the bath; exposures to various NPPB concentrations (brown bars) in the continued presence of 80 mM MOPS&#e032; (green bar; magnified in yellow inset). Login to comment 117 ABCC7 p.Lys1250Ala (E) Macroscopic K1250A CFTR currents at &#e032;40 mV elicited by brief exposures to 10 mM ATP in the absence or presence of blockers. Current relaxations after ATP removal were fitted by single exponentials with time constants indicated. Login to comment 118 ABCC7 p.Lys1250Ala (F) Macroscopic K1250A closing rates in the absence of blocker (gray) and in the presence of 100 &#b5;M NPPB (brown), 80 mM MOPS&#e032; (green), or 100 &#b5;M NPPB + 80 mM MOPS&#e032; (striped). Login to comment 121 ABCC7 p.Lys1250Ala In the presence of 100 &#b5;M NPPB, K1250A closing rate was accelerated by approximately twofold (Fig. 2, E [top and bottom traces, brown fit line and time constant] and F [brown bar]). Login to comment 122 ABCC7 p.Lys1250Ala In contrast, 80 mM MOPS&#e032; neither affected K1250A closing rate (Fig. 2, E [top trace, green fit line and time constant] and F [green bar]), nor prevented the accelerating effect of 100 &#b5;M NPPB (Fig. 2, E [bottom trace, second application of NPPB] and F [striped bar]). Login to comment 125 ABCC7 p.Lys1250Ala To dissect potential effects of 3NB (the NPPB head) and 3PP (the NPPB tail) on permeation and gating, we first characterized effects on permeation using locked-open macroscopic K1250A CFTR currents at two different voltages (&#e032;80 and 60 mV). Login to comment 126 ABCC7 p.Lys1250Ala As expected, 3NB, which contains the pore-blocking carboxylate, dose-dependently suppressed currents through locked-open K1250A channels, and pore block was more pronounced at negative voltages, attesting to its voltage dependence (Fig. 3, A and B). Login to comment 130 ABCC7 p.Lys1250Ala (A, B, F, G, I, and J) Decaying macroscopic currents of locked-open K1250A CFTR channels after removal of ATP, recorded at membrane potentials of &#e032;80 (A, F, and I) or 60 mV (B, G, and J) and responses to brief applications of various concentrations of 3NB (A and B, blue bars), 3PP-sulfate (F and G, red bars), or sulfate (I and J, green bars). Login to comment 135 ABCC7 p.Lys1250Ala (D) Responses of decaying macroscopic locked-open K1250A CFTR current to brief applications of 32 mM 3NB (blue bars) at various membrane potentials. Login to comment 142 ABCC7 p.Lys1250Ala Thus, using macroscopic locked-open K1250A CFTR currents elicited at either &#e032;80 (Fig. 4, A, C, and E) or 60 mV (Fig. 4, B, D, F), we compared fractional effects of coapplying 32 mM 3NB with 210 &#b5;M NPPB (Fig. 4, A and B, blue and brown bars), 20 mM 3PP with 210 &#b5;M NPPB (Fig. 4, C and D, red and brown bars), or 32 mM 3NB with 20 mM 3PP (Fig. 4, E and F, blue and red bars) with the fractional effects of the same three compounds when applied in isolation at the respective concentrations. Login to comment 151 ABCC7 p.Lys1250Ala Indeed, at positive voltages only a small (&#e07a;10%) enhancement (rather than impairment) of the rate of ion flow through locked-open K1250A channels was observed at high 3PP concentrations (Fig. 3 G); a tentative fit to its dose-response curve yielded a K1/2 of &#e07a;10 mM (Fig. 3 H, red-yellow symbols and red fit line). Login to comment 154 ABCC7 p.Lys1250Ala Indeed, at &#e032;80 mV, exposure of locked-open K1250A CFTR channels to sulfate caused substantial pore block (Fig. 3 I) with similarly anomalous dose dependence, yielding maximal block at &#e07a;5 mM sulfate (Fig. 3 H, green-cyan symbols and green abscissa). Login to comment 167 ABCC7 p.Lys1250Ala (A-F) Responses of decaying macroscopic locked-open K1250A CFTR currents, recorded at membrane potentials of &#e032;80 (A, C, and E) or 60 mV (B, D, and F), to brief exposures to the following drug combinations: (A and B) 32 mM 3NB (blue bars) and/ or 210 &#b5;M NPPB (brown bars), (C and D) 20 mM 3PP (red bars) and/or 210 &#b5;M NPPB (brown bars), and (E and F) 32 mM 3NB (blue bars) and/or 20 mM 3PP (red bars). Login to comment 219 ABCC7 p.Lys1250Ala The effects on average unitary conductance of WT CFTR, as observed in heavily (at 50 Hz) filtered current traces (Fig. 7 A), were consistent with the predictions of the macroscopic pore-block assays performed on locked open K1250A channels (Fig. 3, C and H). Login to comment 228 ABCC7 p.Lys1250Ala To test this, we compared fractional effects of 3NB on steady-state macroscopic (I/Icontrol) and average unitary (i/icontrol) K1250A currents by applying 32 mM 3NB for extended time periods to channels gating at steady-state (in 10 mM ATP; Fig. 8 D, first and second 3NB applications) or briefly to locked-open channels after ATP removal (Fig. 8 D, third 3NB application, expanded in inset; this maneuver measures i/icontrol; compare with Fig. 3). Login to comment 229 ABCC7 p.Lys1250Ala (Note that to increase the success rate of very long recordings, all experiments on K1250A gating shown in Fig. 8 were performed at &#e032;20 mV.) Login to comment 230 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Both application and removal of 3NB to K1250A channels, which are gating at steady-state, evoked simple monophasic current responses (Fig. 8 D; in contrast with Fig. 5 A), and the fractional current reduction under such conditions (Fig. 8 F, left gray bar) was well matched by the fractional effect on ATP removal in patches containing K1250A CFTR channels; the K1250A mutation (Fig. 8 C, cartoon, red stars) disrupts ATP hydrolysis (Fig. 8 C, cartoon, red cross; compare with Ramjeesingh et al. [1999]). Login to comment 231 ABCC7 p.Lys1250Ala Indeed, the presence of 32 mM 3NB accelerated K1250A closing rate by two- to threefold (Fig. 8, A [blue vs. gray fit lines and time constants] and C [blue vs. gray bar]), to a similar extent as reported for NPPB (Fig. 8 C, brown bar; replotted from Csan&#e1;dy and T&#f6;r&#f6;csik [2014]). Login to comment 232 ABCC7 p.Lys1250Ala In contrast, 20 mM 3PP, which did not significantly stimulate WT CFTR opening rate (Fig. 7 E), accelerated K1250A closing rate only slightly, by &#e07a;20% (Fig. 8, B and C [red vs. gray bar]). Login to comment 233 ABCC7 p.Lys1250Ala If 3NB indeed acted as a catalyst for the C1࢒O1 step, then the equilibrium between those two states (Fig. 8 G, cartoon, purple double arrow), as reflected by the open probability of the K1250A mutant (Keq = Po/(1 &#e032; Po)), Figure 8.ߓ Effects of 3NB and 3PP on gating rates under nonhydrolytic conditions. Login to comment 234 ABCC7 p.Lys1250Ala (A and B) Macroscopic K1250A CFTR currents at &#e032;20 mV, elicited by exposures to 10 mM ATP (gray bars) in the absence of drug or in the presence of either 32 mM 3NB (A, blue bar) or 20 mM 3PP (B, red bar). Login to comment 237 ABCC7 p.Lys1250Ala The K1250A mutation (cartoon, red stars) disrupts ATP hydrolysis in site 2 (red cross). Login to comment 238 ABCC7 p.Lys1250Ala (D and E) Macroscopic K1250A CFTR currents elicited by 10 mM ATP at &#e032;20 mV and prolonged exposures to 32 mM 3NB (D, blue bars) or 20 mM 3PP (E, red bars) of channels gating at steady-state. Login to comment 241 ABCC7 p.Lys1250Ala (F) Fractional K1250A CFTR currents at &#e032;20 mV in 32 mM 3NB (left pair of bars) or 20 mM 3PP (right pair of bars) applied during steady-state gating (gray bars) or in the locked-open state (yellow bars). Login to comment 243 ABCC7 p.Lys1250Ala Fractional effects on Po for K1250A CFTR were calculated as in Fig. 5 (D and H). Login to comment 252 ABCC7 p.Lys1250Ala Similarly, the small fractional effect of 20 mM 3PP on steady-state K1250A currents (Fig. 8, E and F [right gray bar]) was well explained by a similar small fractional increase in unitary conductance at this voltage (Fig. 8 F, right yellow bar), revealing no change in Po (Fig. 8 G, red bar). Login to comment 343 ABCC7 p.Gly551Asp A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. Login to comment