ABCMdb

PMID: 20628841

Shimizu H, Yu YC, Kono K, Kubota T, Yasui M, Li M, Hwang TC, Sohma Y
A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.
J Physiol Sci. 2010 Sep;60(5):353-62. doi: 10.1007/s12576-010-0102-2. Epub 2010 Jul 14., [PubMed]

Sentences

No. Mutations Sentence Comment

16 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Contrary to WT-CFTR, CFTR mutants whose ATP hydrolysis at NBD2 is abolished (i.e., E1371S, K1250A), can remain open for minutes [9, 12, 17-20]. Login to comment 19 ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe The degree of dose-response shift, a descending order of Y1218G, Y1219I, Y1219F and Y1219W, seemed to depend on the similarity of chemical properties, e.g., hydrophobicity and p bond by aromatic ring, of the side chains introduced by the mutations to tyrosine. Login to comment 21 ABCC7 p.Tyr1219Gly However, interestingly, in macroscopic currents obtained from inside-out patch expressing Y1219G-CFTR, we have observed a significant slow component in the current relaxation after ATP washout. Login to comment 22 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly This suggests that a fraction of Y1219G-CFTR channels in the patch exhibited long-lasting openings after ATP washout whereas the remaining majority of Y1219G-CFTR channels closed within hundreds of milliseconds as WT-CFTR. Login to comment 57 ABCC7 p.Tyr1219Gly We next examined the macroscopic current relaxation in inside-out patches containing those mutants. Indeed, the current decay upon washout of ATP for the Y1219G mutants consistently followed a biphasic time course with a visible slow component (Fig. 2a). Login to comment 58 ABCC7 p.Trp401Gly In contrast, the current relaxation for the W401G mutants showed a major monotonic fast decay which was followed by some minimal residual channel activity (Fig. 2a). Login to comment 59 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe Figure 2b compares macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W mutants. Login to comment 60 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile Although a slow phase of current decay can be seen with Y1219G and Y1219I, this second phase is hardly discernable for the Y1219W mutant. Login to comment 61 ABCC7 p.Tyr1219Phe The result with the Y1219F mutant lies c4; = 714 ms A B 2 s 0.4 pA ATP 5 mM ATP 5 mM 5 s 40 pA 1 s 20 pA Fig. 1 Macroscopic and microscopic currents of wild-type (WT)-CFTR. Login to comment 65 ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile b A representative current trace of WT-CFTR channel showing immediate closing upon a rapid removal of ATP somewhere between Y1219W and Y1219I. Login to comment 67 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe The time constants of the initial fast phase for all the Y1219 mutants are within hundreds of milliseconds (sfast: 504 &#b1; 76 ms, n = 5 for Y1219G; 450 &#b1; 44 ms, n = 7 for Y1219I; 571 &#b1; 142 ms, n = 4 for Y1219F; 513 &#b1; 66 ms, n = 5 for Y1219W) and there is not significant difference in the fast time constant among these Y1219 mutants (Fig. 2b). Login to comment 68 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe On the other hand, the time constants of the slow component for Y1219F and Y1219W are shorter than those of Y1219G and Y1219I (Fig. 3a). Login to comment 69 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe More importantly, the fraction of the slow component (Fig. 3b) shows a gradual decrease as the mutation becomes less 'preserved` (Y1219G [ Y1219I [ Y1219F [ Y1219W). Login to comment 70 ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Phe It should be noted that, for technical reasons (detailed in ''Discussion``), our calculation of the fraction of the slow component is somewhat overestimated for Y1219F and Y1219W. Login to comment 71 ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Phe The actual values for Y1219F and Y1219W are lower than those shown in Fig. 3b. Login to comment 76 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Phe The high affinity of P-ATP was suggested to come from its slow dissociation rate from NBD1 and NBD2 whereas the closing rate in P-ATP was just a little slower than that in B A C Y1219G ATP 5 mM 10 s 1 pA 25 s 2 pA Y1219G 25 s 2 pA ATP 5 mM W401G W 9 1 2 1 Y I 9 1 2 1 Y Y1219F 50 s 50 s 20 pA 50 s 20 pA 50 s 20 pA 20 pA ATP 5 mM 10 s 0.4 pA Y1219G Fig. 2 Macroscopic current relaxations for Y1219 and W401 mutants. Login to comment 77 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly a Representative traces of Y1219G and W401G macroscopic currents responding to a rapid application and removal of 5 mM ATP. Login to comment 78 ABCC7 p.Tyr1219Gly The extended trace marks part of the slow current decay of Y1219G-CFTR after ATP removal. Login to comment 80 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe b Macroscopic current relaxations upon ATP wash-out for Y1219G, Y1219I, Y1219F and Y1219W. Login to comment 82 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe ABCC7 p.Tyr1219Phe ABCC7 p.Tyr1219Phe The time constant and the fraction of the slow component are summarized in Fig. 3. c Single channel recording of Y1219G-CFTR showing a long opening even after ATP removal 35 30 25 20 15 10 5 0 Y1219G Y1219I Y1219F Y1219W 0.5 0.4 0.3 0.2 0.1 0.0 Y1219G Y1219I Y1219F Y1219W A slow / (A slow + A fast ) c4; slow (s) A B Fig. 3 Kinetic parameters of macroscopic current relaxations for Y1219G, Y1219I, Y1219F and Y1219W. Login to comment 83 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe a Time constants of the slow component of the current relaxation for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5. b Fraction of the slow component (Aslow/Aslow ? Login to comment 84 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe Afast) for the Y1219 mutants. Y1219G: n = 5; Y1219I: n = 7; Y1219F: n = 4; and Y1219W: n = 5 ATP because of their similar rate-limiting hydrolyzing rates [11]. Login to comment 85 ABCC7 p.Tyr1219Ile Figure 4 shows the effects of application and removal of 5 mM ATP and 100 lM P-ATP on the macroscopic current of the Y1219I mutant. Login to comment 92 ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Ile Since the slow phase of current decay in Y1219I-CFTR channels was diminished when opened by P-ATP (Fig. 4a), we next examined the accessibility of P-ATP to the binding site during the long-lasting open state by testing if P-ATP can rapidly close Y1219I channels in the long-lasting open state. Login to comment 93 ABCC7 p.Tyr1219Ile We first opened Y1219I-CFTR channels with ATP. Login to comment 106 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Since the life time of these long-lasting openings (in tens of seconds) is very similar to that of hydrolysis-deficient CFTR mutants such as K1250A and E1371S [9, 12, 17-20], it seems difficult to explain these events with the conventional theory that ATP hydrolysis closes the channel. Login to comment 109 ABCC7 p.Tyr1219Ile We show a correlation between the occurrence of these events and the degree of P-ATP 100 bc;M ATP 5 mM P-ATP 100 bc;M P-ATP 100 bc;M A B 50 s 20 pA 10 s 5 pA ATP 5 mM Fig. 4 Effects of P-ATP on Y1219I mutant. Login to comment 110 ABCC7 p.Tyr1219Ile a A representative macroscopic current trace of Y1219I-CFTR responding to rapid application and removal of 5 mM ATP or 100 lM P-ATP. Login to comment 112 ABCC7 p.Tyr1219Ile b Effects of a brief rapid application of P-ATP on Y1219I macroscopic current during the slow phase of the current relaxation after ATP wash-out. Login to comment 118 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser On the other hand, CFTR channels can open for hundreds of seconds when ATP hydrolysis is abolished by mutations such as E1371S or K1250A [9, 12, 17-20]. Login to comment 121 ABCC7 p.Trp401Gly In contrast, decreasing ATP binding in NBD1 by the W401G mutation does not induce a significant slow component of current relaxations (Fig. 2a). Login to comment 132 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe Although the results with Y1219G and Y1219I mutations are quantified accurately, this may not be the case with the data for Y1219F and Y1219W. Login to comment 133 ABCC7 p.Tyr1219Trp First, like WT-CFTR, the slow component for Y1219W is too obscure for reliable curve fitting. Login to comment 135 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile This effect is expected to be small when the probability of the stable open state is high such as in Y1219G or Y1219I. Login to comment 139 ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Ile Since P-ATP has a [50-fold higher affinity to NBD2 compared with ATP [11], P-ATP may also assume a high affinity for the Y1219 mutants. Indeed, while we need millimolar ATP to elicit significant currents from Y1219I-CFTR, 100 lM P-ATP can generate a current similar to the initial current induced by 5 mM ATP (Fig. 4a), indicating that P-ATP is more potent in opening Y1219I channels. Login to comment 140 ABCC7 p.Tyr1219Ile Thus, the high-affinity ATP analog, P-ATP, can effectively abolish the stable open state of Y1219I-CFTR, corroborating the idea that weakening ATP binding at NBD2 promotes the stable open state. Login to comment 146 ABCC7 p.Tyr1219Ile Although P-ATP is able to shorten the lifetime of ATP-opened Y1219I mutants (Fig. 4a), we could not conclude whether the bound ATP has dissociated so that the binding pocket is now unoccupied or the dislocated ATP is still in the binding pocket. Login to comment 151 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile In fact, even when ATP affinity is drastically reduced by mutations such as Y1219G and Y1219I, the probability of the stable open state remains quite low compared to the short-lived open state. Login to comment 162 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe If different mutations at Y1219 can affect the entry rate and the exit rate differently as shown in Table 2, this hypothesis may explain most of the results Table 1 Parameter sets (s-1 ) for the ''kiss and run`` hypothesis kCO1 kO1C kO1O2 kO2C Y1219G 3 or 0a 3 2 9 10-2 3.8 9 10-2 Y1219I : : 1.3 9 10-2 : Y1219F : : 1.1 9 10-2 : Y1219W : : 4 9 10-3 : a kCO1 was set to 3 s-1 for simulating the ''with ATP`` condition and 0 s-1 for simulating the ''after the ATP washout`` condition. Login to comment 166 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Trp ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe b Representative reproduced macroscopic current relaxations mimicking those in Y1219G (red), Y1219I (green), Y1219F (cyan) and Y1219W (blue). Login to comment 172 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile In addition, the slow time constant for Y1219G is not different from that for Y1219I despite the fraction of the slow phase is different between these two mutants. Login to comment