ABCMdb

PMID: 12034762

Dousmanis AG, Nairn AC, Gadsby DC
Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.
J Gen Physiol. 2002 Jun;119(6):545-59., [PubMed]

Sentences

No. Mutations Sentence Comment

25 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Thus, although ATPase activity is diminished 10-20-fold in mutant K464A CFTR, and practically abolished in K1250A CFTR (Ramjeesingh et al., 1999), channel opening rate at millimolar [MgATP] has been reported to be reduced only 2-4-fold in K464A and somewhat more severely (5-10-fold) in K1250A CFTR (Carson et al., 1995; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999); and gating persists even in double mutant K464A/K1250A CFTR channels (Carson et al., 1995). Login to comment 29 ABCC7 p.Lys1250Ala The finding that K1250A CFTR channels, mutated within the NBD2 catalytic site, when exposed to millimolar MgATP alone displayed prolonged open bursts comparable to those elicited by MgAMPPNP in wild-type channels, suggested that NBD2 comprises the active site that controls normal termination of open bursts (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment 31 ABCC7 p.Lys1250Ala A possibly related finding is that the open burst duration of K1250A CFTR channels, though prolonged at millimolar MgATP, has been reported to be brief at micromolar MgATP (Zeltwanger et al., 1999; Ikuma and Welsh, 2000). Login to comment 136 ABCC7 p.Lys1250Ala This is consonant with earlier conclusions that ATP hydrolysis prompts channel closure, based on extreme stabilization of the open state of WT CFTR channels exposed to MgATP plus a poorly hydrolyzable analogue (like MgAMPPNP; see below), or of K1250A mutant channels exposed to just MgATP (Hwang et al., 1994; compare with Gunderson and Kopito, 1994, 1995; Carson et al., 1995). Login to comment 195 ABCC7 p.Lys1250Ala The observation that mutant K1250A CFTR channels show similarly prolonged bursts during exposure to MgATP alone suggested that NBD2 contains the catalytic site where hydrolysis leads to channel closure (Gunderson and Kopito, 1994, 1995; Carson et al., 1995; Zeltwanger et al., 1999). Login to comment 196 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Analysis of the temporal asymmetry of changes in character of rapid current blocking events during open bursts of CFTR channels, and their modification by nucleotide analogues and by mutation of NBD2 (K1250A) but not NBD1 (K464A), provided additional evidence that hydrolysis of ATP tightly bound at NBD2 causes the channel to close (Gunderson and Kopito, 1995). Login to comment 202 ABCC7 p.Lys1250Ala Presumably, when hydrolysis is prevented, either by lack of Mg2ϩ ions (Li et al., 1996) or by NBD mutation (e.g., K1250A; Ramjeesingh et al., 1999), dissociation of nonhydrolyzed nucleotide from NBD2 becomes the rate-limiting step for the delayed channel closure. Login to comment 246 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Met Nucleotide binding assays (at 0ЊC to prevent hydrolysis) using 8-azidoATP photolabeling show that binding occurs with the same micromolar apparent affinity in wild-type, mutant K1250M, and double mutant K464A/ K1250A, CFTR (Carson et al., 1995). Login to comment 247 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala These findings, together with the fact that even double mutant K464A/ K1250A CFTR channels open and close at measurable rates (Carson et al., 1995), make it seem unlikely that ATP hydrolysis at either NBD1 or NBD2 is a prerequisite for channel opening. Login to comment 372 ABCC7 p.Gly551Asp ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. Login to comment