ABCMdb

PMID: 7543023

Gunderson KL, Kopito RR
Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis.
Cell. 1995 Jul 28;82(2):231-9., [PubMed]

Sentences

No. Mutations Sentence Comment

57 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Met ABCC7 p.Lys1250Thr ABCC7 p.Lys1250Gly These NBF1 and NBF2 mutants, harboring the individual mutations K464A and K1250A, K1250G, K1250M, or K1250T, respectively, were expressed in HEK cells and reconstituted into planar lipid bilayers from which single-channel currents were recorded (Figures 3A and 3B). Login to comment 59 ABCC7 p.Lys464Ala Compared with wild-type CFTR, the NBF1 mutant K464A exhibited slowed gating kinetics and reduced open probability (Po) in the presence of 1 mM MgATP (Figure 3A; see Figure 5). Login to comment 68 ABCC7 p.Lys1250Met The mean conductance of K1250M channels was 8.9 __. 0.13 (n = 4) pS (Figures 3C and 3D), which is not significantly different from the conductance of the wild-type O1 state. Our data show that the O~--*O= transition is highly asymmetric in wild-type channels, consistent with the large free energy change associated with ATP hydrolysis. Login to comment 69 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala We propose that A -PPi +PPi • - 0[ •; K1250A KI250A . Login to comment 70 ABCC7 p.Lys1250Met c KI250G ' C K1250M - :~:-~ ~ . Login to comment 74 ABCC7 p.Lys464Ala Effect of Mutation of the P Loop Lysine in NBF1 and NBF2 on CFTR Gating (A) Single-channel record of K464A in the absence (top) or presence (bottom)of 2 mM PP~. Login to comment 76 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Met ABCC7 p.Lys1250Thr ABCC7 p.Lys1250Gly (B)Single-channelrecordsof K1250A,K1250G, K1250M, and K1250T filtered at 50 Hz under standard cis bath conditions. Login to comment 79 ABCC7 p.Lys1250Ala Mean conductance values are 9.0 _+ 0.19 pS (n = 4) for the O1state; 10.3 _+0.20 pS (n = 4) for the 02 state; and 8.9 _+ 0.13 pS (n = 4) for the single conductance state of the K1250A mutant. Login to comment 81 ABCC7 p.Lys1250Ala (D) Single-channel recording of K1250A mutant at different holding potentials used in generating the I-V curve shown above. Login to comment 92 ABCC7 p.Asp1370Asn 0 2 B low MI 2* C D1370N (high Mg2+) 2sec -----]0,5 pA Figure 4. Login to comment 109 ABCC7 p.Asp1370Asn Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment 110 ABCC7 p.Asp1370Asn Gating of CFTR channels harboring a D1370N mutation closely resembled that of the wild-type channel in EDTA (Figure 4C). Login to comment 111 ABCC7 p.Asp1370Asn Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment 112 ABCC7 p.Asp1370Asn Moreover, even in the presence of Mg2+,we were unable to induce locking of the D1370N channels with PPi (data not shown). Login to comment 114 ABCC7 p.Asp1370Asn By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2÷, and a subsequent state that requires Mg2÷ and is effectively irreversible. Login to comment 115 ABCC7 p.Asp1370Asn By contrast, D1370N mutants, which can be mimicked by applying EDTA to wild-type channels, are also blocked from completing the full (C~O1~O2~C) gating cycle through 02, but are neither locked open in O1 nor capable of becoming locked by PP~.These two classes of mutants thus define two kinetic states of the O1 conductance level: an initial open state that is reversible (back to C) and independent of Mg2&#f7;, and a subsequent state that requires Mg2&#f7; and is effectively irreversible. Login to comment 117 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The effect of this double mutation, G1247D and G1249E, is to introduce negative charge into the P loop, which should severely attenuate nucleotide binding to NBF2. Login to comment 118 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The effect of this double mutation, G1247D and G1249E, is to introduce negative charge into the P loop, which should severely attenuate nucleotide binding to NBF2. Login to comment 121 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment 122 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o ~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment 178 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The proposal that ATP binding to NBF2 opens the channel to the O1 state is supported by the virtual absence of ATP-dependent openings in the G1247D, G1249E P loop mutant. Login to comment 179 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The proposal that ATP binding to NBF2 opens the channel to the O1 state is supported by the virtual absence of ATP-dependent openings in the G1247D, G1249E P loop mutant. Login to comment 214 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Thr ABCC7 p.Lys1250Gly Polymerase Chain Reaction Megaprimer Mutagenesis The following site-directed mutants were constructed by using the megaprimer polymerase chain reaction (PCR)-based mutagenesis protocol (Landt et al., 1990; Sarkar and Sommer, 1990): K464A, K1250A, K1250G, K1250T, and GG1247, 1249DE. Login to comment 215 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Thr ABCC7 p.Lys1250Gly Polymerase Chain Reaction Megaprimer Mutagenesis The following site-directed mutants were constructed by using the megaprimer polymerase chain reaction (PCR)-based mutagenesis protocol (Landt et al., 1990; Sarkar and Sommer, 1990): K464A, K1250A, K1250G, K1250T, and GG1247, 1249DE. Login to comment 241 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment 242 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment