scholarly journals Modulation of CFTR gating by permeant ions

2014 ◽  
Vol 145 (1) ◽  
pp. 47-60 ◽  
Author(s):  
Han-I Yeh ◽  
Jiunn-Tyng Yeh ◽  
Tzyh-Chang Hwang
Keyword(s):  
Atp Hydrolysis ◽  
Single Channel ◽  
Common Mechanism ◽  
Transmembrane Conductance Regulator ◽  
Open Probability ◽  
Binding Domains ◽  
Gating Model ◽  
Sites Of Action ◽  
Opening And Closing ◽  
Closing Rate

Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR’s transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (ΔNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

scholarly journals A common mechanism for CFTR potentiators

2017 ◽  
Vol 149 (12) ◽  
pp. 1105-1118 ◽  
Author(s):  
Han-I Yeh ◽  
Yoshiro Sohma ◽  
Katja Conrath ◽  
Tzyh-Chang Hwang
Keyword(s):  
Cystic Fibrosis ◽  
Atp Hydrolysis ◽  
Single Channel ◽  
Underlying Mechanism ◽  
Common Mechanism ◽  
Loss Of Function ◽  
Open Probability ◽  
High Throughput Drug Screening ◽  
State Dependent ◽  
Opening And Closing

Cystic fibrosis (CF) is a channelopathy caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a phosphorylation-activated and adenosine triphosphate (ATP)–gated chloride channel. In the past few years, high-throughput drug screening has successfully realized the first US Food and Drug Administration–approved therapy for CF, called ivacaftor (or VX-770). A more recent CFTR potentiator, GLPG1837 (N-(3-carbamoyl-5,5,7,7-tetramethyl-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-3-carboxamide), has been shown to exhibit a higher efficacy than ivacaftor for the G551D mutation, yet the underlying mechanism of GLPG1837 remains unclear. Here we find that despite their differences in potency and efficacy, GLPG1837 and VX-770 potentiate CFTR gating in a remarkably similar manner. Specifically, they share similar effects on single-channel kinetics of wild-type CFTR. Their actions are independent of nucleotide-binding domain (NBD) dimerization and ATP hydrolysis, critical steps controlling CFTR’s gate opening and closing, respectively. By applying the two reagents together, we provide evidence that GLPG1837 and VX-770 likely compete for the same site, whereas GLPG1837 and the high-affinity ATP analogue 2′-deoxy-N6-(2-phenylethyl)-adenosine-5′-O-triphosphate (dPATP) work synergistically through two different sites. We also find that the apparent affinity for GLPG1837 is dependent on the open probability of the channel, suggesting a state-dependent binding of the drug to CFTR (higher binding affinity for the open state than the closed state), which is consistent with the classic mechanism for allosteric modulation. We propose a simple four-state kinetic model featuring an energetic coupling between CFTR gating and potentiator binding to explain our experimental results.

scholarly journals Electrostatic tuning of the pre- and post-hydrolytic open states in CFTR

2017 ◽  
Vol 149 (3) ◽  
pp. 355-372 ◽  
Author(s):  
Jingyao Zhang ◽  
Tzyh-Chang Hwang
Keyword(s):  
Atp Hydrolysis ◽  
Single Channel ◽  
Differential Sensitivity ◽  
Transmembrane Conductance Regulator ◽  
Net Charge ◽  
Channel Current ◽  
Binding Domains ◽  
Similar Phenotype ◽  
Conductance Changes ◽  
Open States

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that couples adenosine triphosphate (ATP) hydrolysis at its nucleotide-binding domains to gating transitions in its transmembrane domains. We previously reported that the charge-neutralized mutant R352C shows two distinct open states, O1 and O2. The two states could be distinguished by their single-channel current amplitudes: O1 having a smaller amplitude (representing a prehydrolytic open state) and O2 having a larger amplitude (representing a post-hydrolytic open state). In this study, a similar phenotype is described for two mutations of another pore-lining residue, N306D and N306E, suggesting that alterations of the net charge within CFTR’s pore confer this unique conductance aberration. Because moving either of the two endogenous charges, R303 and R352, to positions further along TM5 and TM6, respectively, also results in this O1O2 phenotype, we conclude that the position of the charged residue in the internal vestibule affects hydrolysis-dependent conductance changes. Furthermore, our data show that the buffer and CFTR blocker morpholino propane sulfonic acid (MOPS−) occludes the O1 state more than it does the O2 state when the net charge of the internal vestibule is unchanged or increased. In contrast, when the net charge in the internal vestibule is decreased, the differential sensitivity to MOPS− block is diminished. We propose a three-state blocking mechanism to explain the charge-dependent sensitivity of prehydrolytic and post-hydrolytic open states to MOPS− block. We further posit that the internal vestibule expands during the O1 to O2 transition so that mutation-induced electrostatic perturbations within the pore are amplified by the smaller internal vestibule of the O1 state and thus result in the O1O2 phenotype and the charge-dependent sensitivity of the two open states to MOPS− block. Our study not only relates the O1O2 phenotype to the charge distribution in CFTR’s internal vestibule but also provides a toolbox for mechanistic studies of CFTR gating by ATP hydrolysis.

scholarly journals Cysteine accessibility probes timing and extent of NBD separation along the dimer interface in gating CFTR channels

2015 ◽  
Vol 145 (4) ◽  
pp. 261-283 ◽  
Author(s):  
Luiz A. Poletto Chaves ◽  
David C. Gadsby
Keyword(s):  
Atp Hydrolysis ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Dimer Interface ◽  
Binding Domains ◽  
Channel Opening ◽  
Order Of Magnitude ◽  
Signature Sequence ◽  
Dependent Modification ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR) channel opening and closing are driven by cycles of adenosine triphosphate (ATP) binding–induced formation and hydrolysis-triggered disruption of a heterodimer of its cytoplasmic nucleotide-binding domains (NBDs). Although both composite sites enclosed within the heterodimer interface contain ATP in an open CFTR channel, ATP hydrolysis in the sole catalytically competent site causes channel closure. Opening of the NBD interface at that site then allows ADP–ATP exchange. But how frequently, and how far, the NBD surfaces separate at the other, inactive composite site remains unclear. We assessed separation at each composite site by monitoring access of nucleotide-sized hydrophilic, thiol-specific methanothiosulfonate (MTS) reagents to interfacial target cysteines introduced into either LSGGQ-like ATP-binding cassette signature sequence (replacing equivalent conserved serines: S549 and S1347). Covalent MTS-dependent modification of either cysteine while channels were kept closed by the absence of ATP impaired subsequent opening upon ATP readdition. Modification while channels were opening and closing in the presence of ATP caused macroscopic CFTR current to decline at the same speed as when the unmodified channels shut upon sudden ATP withdrawal. These results suggest that the target cysteines can be modified only in closed channels; that after modification the attached MTS adduct interferes with ATP-mediated opening; and that modification in the presence of ATP occurs rapidly once channels close, before they can reopen. This interpretation was corroborated by the finding that, for either cysteine target, the addition of the hydrolysis-impairing mutation K1250R (catalytic site Walker A Lys) similarly slowed, by an order of magnitude, channel closing on ATP removal and the speed of modification by MTS reagent in ATP. We conclude that, in every CFTR channel gating cycle, the NBD dimer interface separates simultaneously at both composite sites sufficiently to allow MTS reagents to access both signature-sequence serines. Relatively rapid modification of S1347C channels by larger reagents—MTS-glucose, MTS-biotin, and MTS-rhodamine—demonstrates that, at the noncatalytic composite site, this separation must exceed 8 Å.

Control of the CFTR channel's gates

2005 ◽  
Vol 33 (5) ◽  
pp. 1003-1007 ◽  
Author(s):  
P. Vergani ◽  
C. Basso ◽  
M. Mense ◽  
A.C. Nairn ◽  
D.C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Ion Channel ◽  
Single Channel ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Binding Domains ◽  
Abc Protein ◽  
Channel Opening ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1–NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

scholarly journals Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

2010 ◽  
Vol 135 (5) ◽  
pp. 399-414 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Min Li ◽  
Tzyh-Chang Hwang
Keyword(s):  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Abc Proteins ◽  
Atp Binding Site ◽  
Functional Studies ◽  
Binding Domains ◽  
Gating Model ◽  
Atp Binding And Hydrolysis ◽  
Opening And Closing

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

scholarly journals Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation

2012 ◽  
Vol 140 (4) ◽  
pp. 347-359 ◽  
Author(s):  
Kang-Yang Jih ◽  
Yoshiro Sohma ◽  
Tzyh-Chang Hwang
Keyword(s):  
Conformational Change ◽  
Atp Hydrolysis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
External Energy ◽  
Binding Domains ◽  
Abc Protein ◽  
Gating Model ◽  
Conductance States ◽  
Pore Lining

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR’s gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating—a strict coupling between the ATP hydrolysis cycle and the gating cycle—is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1→O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1→O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

scholarly journals Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

2000 ◽  
Vol 116 (3) ◽  
pp. 477-500 ◽  
Author(s):  
László Csanády ◽  
Kim W. Chan ◽  
Donna Seto-Young ◽  
David C. Kopsco ◽  
Angus C. Nairn ◽  
...  
Keyword(s):  
Single Channel ◽  
Tight Binding ◽  
Nucleotide Binding ◽  
Open Probability ◽  
Binding Domains ◽  
Highly Active ◽  
Level Of Activity ◽  
Strong Stimulation ◽  
Opening And Closing ◽  
R Domain

Opening and closing of a CFTR Cl− channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634–836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1–633 and 837–1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

Mg2+-dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2)

2008 ◽  
Vol 416 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Luba Aleksandrov ◽  
Andrei Aleksandrov ◽  
John R. Riordan
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bivalent Cation ◽  
Binding Domains ◽  
Rapid Hydrolysis ◽  
Cystic Fibrosis Transmembrane

ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419–15425]. Subsequent to the initial binding, Mg2+ drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497–500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.

Functional analysis and molecular model of the human urate transporter/channel, hUAT

2002 ◽  
Vol 283 (1) ◽  
pp. F150-F163 ◽  
Author(s):  
Edgar Leal-Pinto ◽  
B. Eleazar Cohen ◽  
Michael S. Lipkowitz ◽  
Ruth G. Abramson
Keyword(s):  
Functional Analysis ◽  
Lipid Bilayers ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Molecular Model ◽  
Open Probability ◽  
Homologous Proteins ◽  
Urate Transporter ◽  
Binding Domains

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two β-galactoside binding domains that bind lactose. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.

Mechanism of activation of Xenopus CFTR by stimulation of PKC

2004 ◽  
Vol 287 (5) ◽  
pp. C1256-C1263 ◽  
Author(s):  
Yongyue Chen ◽  
Guillermo A. Altenberg ◽  
Luis Reuss
Keyword(s):  
Single Channel ◽  
Membrane Conductance ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Substituted Cysteine Accessibility ◽  
Maximal Stimulation ◽  
A Minor ◽  
Cystic Fibrosis Transmembrane ◽  
Stimulation Of

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR ( XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ∼90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (∼30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

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