A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

Hennager, Daniel J.; Ikuma, Mutsuhiro; Hoshi, Toshinori; Welsh, Michael J.

doi:10.1073/pnas.051633298

Cited by 15 publications

(15 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In such a situation, there is no reason to count transitions and refer to the mismatch as evidence of nonequilibrium as most of the transitions have been lost already. More precise kinetic analysis with extended bandwidth does not reveal any evidence of nonequilibrium in CFTR ion channel gating [27]. This confirms our analysis of the temperature dependence of gating transitions which revealed that they were indeed in thermodynamic equilibrium [3].…”

Section: Is There Thermal Nonequilibrium In Cftr Channel Gating?supporting

confidence: 76%

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Aleksandrov

Riordan

2006

Pflugers Arch - Eur J Physiol

View full text Add to dashboard Cite

As the product of the gene mutated in cystic fibrosis, the most common genetic disease of Caucasians, CFTR is an atypical ABC protein. From an evolutionary perspective, it is apparently a relatively young member of the ABC family, present only in metazoans where it plays a critical role in epithelial salt and fluid homeostasis. Functionally, the membrane translocation process it mediates, the passive bidirectional diffusion of small inorganic anions, is simpler than the vectorial transport of larger more complex substrates ("allocrites") by most ABC transporters. However, the control of the permeation pathway which cannot go unchecked is necessarily more stringent than in the case of the transporters. There is tight regulation by the phosphorylation/dephosphorylation of the unique CFTR R domain superimposed on the basic ABC regulation mode of ATP binding and hydrolysis at the dual nucleotide binding sites. As with other ABCC subfamily members, only the second of these sites is hydrolytic in CFTR. The phosphorylation and ATP binding/hydrolysis events do not strongly influence each other; rather, R domain phosphorylation appears to enable transduction of the nucleotide binding allosteric signal to the responding channel gate. ATP hydrolysis is not required for either the opening or closing gating transitions but efficiently clears the ligand-binding site enabling a new gating cycle to be initiated.

show abstract

Section: Is There Thermal Nonequilibrium In Cftr Channel Gating?supporting

confidence: 76%

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Aleksandrov

Riordan

2006

Pflugers Arch - Eur J Physiol

View full text Add to dashboard Cite

show abstract

“…In such a model, the burst duration would remain unchanged regardless of whether the channel had opened with ATP bound to both or only a single NBD. Interestingly, CFTR can open infrequently without ATP, and the burst duration in the absence of ATP is only slightly shorter than the burst duration in the presence of ATP (45). Also consistent with this hypothesis are thermodynamic studies that reveal only a small energy barrier to channel closure (47,48), but this energy barrier need not be related to ATP hydrolysis or dissociation.…”

Section: At Nbd1 Atp Binding Plays a More Important Role Than Hydrolsupporting

confidence: 67%

“…It has been unclear what determines the rate at which CFTR closes. In WT CFTR, the rate of channel closing is relatively independent of ATP concentration (19,42,43), although a small effect has been reported (12,20,44,45). These results suggest that ATP binding does not close the channel.…”

Section: At Nbd1 Atp Binding Plays a More Important Role Than Hydrolmentioning

confidence: 49%

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

Berger

Ikuma

Welsh

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [␣-32 P]8-N3-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.cystic fibrosis ͉ ion channel ͉ Walker motif T he cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl Ϫ channel in the ABC transporter family of membrane proteins (1, 2). It contains two membrane-spanning domains that form the channel pore, an R domain through which phosphorylation regulates channel activity, and two highly conserved nucleotide-binding domains (NBD1 and NBD2) that control channel opening and closing through their interaction with ATP.Structural studies have suggested how nucleotides interact with ABC transporter NBDs (3-9). Each NBD contains a conserved phosphate-binding loop (P-loop or Walker A motif) that wraps around the ATP phosphates to form part of an ATP-binding pocket. Each NBD also contains a ''signature'' LSGGQ motif. Some structures suggest that the two NBDs form a dimer clamping two nucleotides at the NBD:NBD interface (7, 10). In the dimeric structures, ATP binds between the P-loop of one NBD and the signature motif of the other NBD. (In this article, we will refer to an NBD1 ATP-binding site as that which involves the P-loop of NBD1, and the NBD2 site as that which involves the P-loop of NBD2.) Our recent findings have provided functional evidence for NBD dimerization (11).Earlier work showed that ATP binds both CFTR NBDs and that the protein hydrolyzes ATP (12-15). Clues to the role that the two individual NBDs play in gating have come f...

show abstract

“…Whether ATP binding to CFTR is essential for channel opening (e.g., by a sequential/strict coupling mechanism) or simply shifts the equilibrium between states (e.g., by an allosteric mechanism) is not entirely clear. Several groups have reported very low activities of WT and certain mutant CFTR channels in the apparent absence of ATP (11,12), which would be consistent with an allosteric gating scheme (13). However, interpreting these results is confounded by uncertainties about the completeness of ATP removal by bath perfusion alone (i.e., if scavenging enzymes are not used) or whether the observed activity represents normal CFTR gating.…”

mentioning

confidence: 76%

ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

Wang

Bernard

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

162

View full text Add to dashboard Cite

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligandgated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.ATP binding cassette transporter | cystic fibrosis | ligand | constitutive | mutant C ystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily, although it is the only known ion channel in this transporter family (1). Like other ABC transporters, CFTR uses ATP binding to its two nucleotide binding domains (NBDs) to drive conformational rearrangements of its transmembrane domains (2, 3). CFTR channel opening is linked to ATP binding to each of two sites at the interface of an NBD1-NBD2 dimer (2, 3). Subsequent hydrolysis, typically at site 2 (primarily composed of sequences from NBD2), promotes channel closure by clearing ligand from this site (4, 5). The coupling between ATP binding and pore opening is presumably mediated by the cytosolic loops that physically link the NBDs to the transmembrane domains (6, 7).Because CFTR is an enzyme that normally hydrolyzes its ligand as part of the channel gating cycle, the extent to which its properties are similar to those of more conventional ligand-gated channels is an interesting issue. Ligand-gated channels such as acetylcholine receptors obey the principles of protein alloster...

show abstract

A conditional probability analysis of cystic fibrosis transmembrane conductance regulator gating indicates that ATP has multiple effects during the gating cycle

Cited by 15 publications

References 24 publications

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

Contact Info

Product

Resources

About