ABCMdb

PMID: 24420771

Csanady L, Torocsik B
Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating.
J Gen Physiol. 2014 Feb;143(2):269-87. doi: 10.1085/jgp.201311089. Epub 2014 Jan 13., [PubMed]

Sentences

No. Mutations Sentence Comment

30 ABCC7 p.Lys1250Ala Recordings were done in the presence of saturating (2 mM) MgATP; for the K1250A mutant 10 mM MgATP was used to compensate for its greatly impaired apparent ATP affinity (Vergani et al., 2003). Login to comment 69 ABCC7 p.Glu1371Ser (E, G, I, and K) Slowly decaying macroscopic "locked-open" currents of E1371S CFTR in the absence of bath ATP; brief applications of 210 &#b5;M NPPB or 20 mM MOPS&#e032; at various voltages (E and G) or of various blocker concentrations at &#e032;120 mV (I and K). Login to comment 84 ABCC7 p.Glu1371Ser A convenient macroscopic pore block assay is afforded by the nonhydrolytic E1371S CFTR mutant, which lacks the catalytic glutamate in site 2. Login to comment 86 ABCC7 p.Glu1371Ser Because they are active in resting cells (compare to Zhou et al., 2010), excision of E1371S multichannel patches into an ATP-free solution typically uncovers large, slowly decaying currents devoid of gating fluctuations (Fig. 1, E, G, I, and K). Login to comment 117 ABCC7 p.Lys1250Ala To test this, we studied the closing rate of K1250A CFTR channels, in which mutation of the NBD2 Walker A lysine abrogates ATP hydrolysis at site 2 (Ramjeesingh et al., 1999) and reduces gating to reversible caused simple monophasic current relaxations (Fig. 3 E), both the addition and removal of NPPB elicited biphasic responses (Fig. 3 D), attesting to the dual effects of this compound. Login to comment 123 ABCC7 p.Lys1250Ala (A) Macroscopic K1250A CFTR current at &#e032;120 mV elicited by exposures to 10 mM ATP in the absence or presence of blockers. Login to comment 126 ABCC7 p.Lys1250Ala The K1250A mutation (B and E, cartoons, red stars) disrupts ATP hydrolysis in site 2 (red cross). Login to comment 127 ABCC7 p.Lys1250Ala (C) Macroscopic K1250A CFTR current elicited by 10 mM ATP at &#e032;40 mV, prolonged exposure to 210 &#b5;M NPPB of channels gating at steady state, and brief exposure to NPPB of surviving locked-open channels after ATP removal (10-s yellow box, expanded in inset). Login to comment 129 ABCC7 p.Lys1250Ala (D) Fractional K1250A CFTR currents at &#e032;40 mV in 210 &#b5;M NPPB applied during steady-state gating (red bar) or in the locked-open state (yellow bar). Login to comment 131 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Fractional effect on Po for K1250A CFTR (blue bar) was calculated as in Fig. 2 E. rate of K1250A upon removal of bath ATP (Fig. 4 A) was &#e07a;100 times slower than for WT channels (Fig. 4, A, red fit line, and B, red bar; compare to Fig. 3, A and C). Login to comment 134 ABCC7 p.Lys1250Ala Consistent with its effect on K1250A closing rate (Fig. 4, A and B), 210 &#b5;M NPPB also accelerated the unlocking rate of pyrophosphate-locked WT CFTR channels by two- to threefold (Fig. S2). Login to comment 135 ABCC7 p.Lys1250Ala Because NPPB accelerated both forward (Fig. 3 F) and backward (Fig. 4 B) transitions of the C1࢒O1 step, we examined whether it affects the equilibrium constant between those two states (Fig. 4 E, cartoon, purple double arrow; Keq), i.e., Po for K1250A channels. Login to comment 136 ABCC7 p.Lys1250Ala Even prolonged exposure to 210 &#b5;M NPPB of K1250A channels gating at steady state in 10 mM ATP (Fig. 4 C; note Vm = &#e032;40 mV) failed to elicit biphasic responses like those seen for WT channels (see Fig. 3 D). Login to comment 141 ABCC7 p.Lys1250Ala Given the observed increase in nonhydrolytic closing rate (Fig. 4, A and B), this result indicates that NPPB must also speed the opening rate of K1250A CFTR, just as it does for WT (Fig. 3 F). Login to comment 160 ABCC7 p.Lys1250Ala (E; left) Macroscopic K1250A CFTR current at +60 mV elicited by exposures to 10 mM ATP in the absence or presence of 210 &#b5;M NPPB; colored lines, single-exponential fits (&#e074;, time constants). Login to comment 161 ABCC7 p.Lys1250Ala (Right) Macroscopic K1250A closing rates (bars, 1/&#e074;) in the absence (red) and presence (blue) of NPPB. Login to comment 176 ABCC7 p.Lys1250Ala Finally, at +60 mV, 210 &#b5;M NPPB increased the nonhydrolytic closing rate of K1250A CFTR by approximately threefold (Fig. 6 E), just as it did at &#e032;120 mV cyan bar). Login to comment 207 ABCC7 p.Glu1371Ser (C) Segments of unitary current at +60 mV from a single locked-open E1371S CFTR channel in symmetrical &#e07a;140-mM Cl&#e032; at three different cytosolic pH values, with and without cytosolically applied 210 &#b5;M NPPB, displayed at two bandwidths (fc, filter corner frequency). Login to comment 217 ABCC7 p.Gly551Asp Recently, various extensions of this scheme have been suggested to explain the rare openings (Po of &#e07a;0.004), in the absence of ATP, of unliganded WT (Szollosi et al., 2010) or G551D CFTR (Bompadre et al., 2007), enhancement of such activity by cytosolic loop mutations (Wang et al., 2010), and [ATP]-dependent prolongation of open burst durations in mutants (Jih et al., 2012b) or by drugs (Jih and Hwang, 2013). Login to comment 218 ABCC7 p.Gly551Asp Although these extended models differ in detail (Szollosi et al., 2010; Kirk and Wang, 2011; Jih et al., 2012a), they all contain, as a common core, the basic cycle depicted in our figure insets, which describes the majority of gating events for both WT CFTR and many CFTR mutants, including &#e044;F508 (Jih et al., 2011; but compare to Wang et al., 2005, for ATP-independent G551D). Login to comment 222 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser In fact, because of its dual action on both i and Po, fractional reduction of macroscopic current by NPPB is not a good measure of pore block, unless observed on "locked-open" channels, which are not gating (Po of &#e07a;1), such as surviving E1371S (Fig. 1, E and I) or K1250A (Fig. 4 D, inset) channels after the removal of ATP. Login to comment 233 ABCC7 p.Gly551Asp However, to date it has been shown to benefit only patients carrying at least one G551D allele, which constitute <5% of the CF population. Login to comment 255 ABCC7 p.Lys1250Ala Accordingly, although the apparent KI of NPPB for pore block was &#e07a;20 &#b5;M at &#e032;120 mV (Fig. 1 J; replotted in Fig. 10, open diamonds), the apparent K1/2 for slowing hydrolytic closure of WT CFTR (Fig. 10, closed circles) was at least four times higher (&#e07a;90 &#b5;M), and a tentative fit to the dose-response curve for acceleration of nonhydrolytic closure (measured for the K1250A mutant; Fig. 10, closed squares) suggested a similar K1/2 of &#e07a;100 &#b5;M (although reliability of the latter fit is limited by the uncertainty of its asymptotic value). Login to comment 259 ABCC7 p.Lys1250Ala Macroscopic closing rates of WT (closed circles) and K1250A (closed squares) CFTR in the presence of various cytosolic [NPPB], measured at &#e032;120 mV using the protocols shown in Figs. 3 A and 4 A, respectively; leftmost data points represent control values in the absence of NPPB. Login to comment 260 ABCC7 p.Lys1250Ala The 50- and 100-&#b5;M data points for K1250A were measured at &#e032;40 mV. Login to comment 261 ABCC7 p.Lys1250Ala Solid lines are fits to the equation rOC([NPPB]) = r0 + (r&#e060; &#e032; r0)([NPPB]/([NPPB] + K1/2)), with r&#e060; fixed to zero for WT but left free for K1250A; K1/2 values are plotted. Login to comment 280 ABCC7 p.Lys1250Ala Accordingly, for the nonhydrolytic K1250A mutant, which gates at equilibrium, NPPB speeds gating transitions but does not alter Po (Fig. 4 E). Login to comment 297 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Third, the presence of NPPB does not slow closure of the nonhydrolytic mutant K1250A (Fig. 4, A and B), confirming that the gate can close with NPPB bound in the pore, just as it can close in the presence of bound MOPS&#e032; , as the latter does not affect the closing rate of either WT (Fig. 3, B and C) or K1250A CFTR (Fig. 4, A and B). Login to comment 302 ABCC7 p.Gly551Asp The potentiator compound Vx-770, now approved for the treatment of CF patients carrying the G551D mutation, was proposed to slow closure of WT CFTR by facilitating a reentry pathway from state O2 to state O1, which would allow ADP in site 2 to be replaced by a new ATP molecule without intervening pore closure (Jih and Hwang, 2013). Login to comment 316 ABCC7 p.Lys1250Ala Gating effects of the K1250A mutation were modeled by setting rate O1࢐O2 to zero while increasing the Kd for ATP to 5 mM (Vergani et al., 2003), those of the &#e044;F508 mutation were modeled by decreasing rate C1࢐O1 30-fold while increasing rate O1࢐C1 threefold (Miki et al., 2010; Jih et al., 2011). Login to comment 320 ABCC7 p.Lys1250Ala ߒ (Fig. 12 A, cube) reduces to the four-state model to its right (blue): apparent rate c6; c6; C O 1 1 ࢐ reflects the observed opening rate in 210 &#b5;M NPPB (Figs. 3 F and 6, B and F), rate c6; c6; O O 1 2 ࢐ is the rate-limiting step for closure and reflects closing rate in 210 &#b5;M NPPB (Figs. 3 C and 6, C and F), c6; c6; O C 2 2 ࢐ seemed unaffected by NPPB (see fits to the distributions of open burst durations in 20 &#b5;M NPPB; Fig. 5 D), whereas rate c6; c6; O C 1 1 ࢐ reflects K1250A closing rate in 210 &#b5;M NPPB (Fig. 4 B). Login to comment 327 ABCC7 p.Lys1250Ala Indeed, the model faithfully reproduced (a) robust stimulation of WT Po as reflected by a discrepancy between fractional effects on steady-state macroscopic and unitary currents (Fig. 12, B and C; compare to Figs. 2, A, B, and E, and 6, A and B) and current over- shoots upon rapid removal of NPPB (Fig. 12 E; compare to Figs. 3 D and 6 D); (b) slowing of WT (hydrolytic) macroscopic closing rate (Fig. 12 D; compare to Figs. 3, A and C, and 6 C); (c) acceleration of WT macroscopic opening rate (Fig. 12 E; compare to Figs. 3 F and 6 D); (d) shortening and prolongation, respectively, of steady-state single-channel mean closed (interburst) and open (burst) durations (Fig. 12, F and H; compare to Figs. 5 B and 6 F), the latter being caused by a slowed rate c6; c6; O O 1 2 ࢐ (Fig. 12 G; compare to Fig. 5, C and D); (e) accelerated closing rate of the nonhydrolytic K1250A mutant (Fig. 12 I; compare to Figs. 4, A and B, and 6 E), but (f) lack of effect on the Po of this channel (Fig. 12 J; compare to Fig. 4, C and D), (g) observed apparent affinities of the NPPB gating effects (Fig. 12 K; compare to Fig. 10), as well as (h) extremely efficient stimulation of low Po mutants such as &#e044;F508 (Fig. 12 L; compare to Fig. 7; also compare to Fig. 8). Login to comment 335 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Finally, rate O1࢐C1 represents the slow rate of nonhydrolytic closure, modeled by the closing rates of nonhydrolytic mutants K1250A (Fig. 4 A) or E1371S (Fig. 1 K), or of WT channels that have been locked open by ATP plus pyrophosphate (Fig. S2); the time constant of this slow process is &#e07a;30 s. Login to comment 354 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 420 ABCC7 p.Gly551Asp A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. Login to comment