ATP-dependent Gating Research Articles

Sulfonylurea receptor coupled conductances alter the performace of two central pattern generating circuits in Cancer borealis.

Neuronal activity and energy supply must maintain a fine balance for neuronal fitness. Various channels of communication between the two could impact network output in different ways. Sulfonylurea receptors (SURs) are a modification of ATP-binding cassette proteins (ABCs) that confer ATP-dependent gating on their associated ion channels. They are widely expressed and link metabolic states directly to neuronal activity. The role they play varies in different circuits, both enabling bursting and inhibiting activity in pathological conditions. The crab, Cancer borealis, has central patterns generators (CPGs) that fire in rhythmic bursts nearly constantly and it is unknown how energy availability influences these networks. The pyloric network of the stomatogastric ganglion (STG) and cardiac ganglion (GC) control rhythmic contractions of the foregut and heart respectively. Pharmacological manipulation of SURs results in opposite effects in the two CPGs. Neuronal firing completely stops in the STG when SUR-associated channels are open, and firing increases when the channels are closed. This results from a decrease in the excitability of pyloric dilator (PD) neurons, which are a part of the pacemaker kernel. The neurons of the CG, paradoxically, increase firing within bursts when SUR-associated channels are opened, and bursting slows when SUR-associated channels are closed. The channel permeability and sensitivities analyses present novel SUR-conductance biophysics, which nevertheless change activity in ways reminiscent of the predominantly studied mammalian receptor/channels. We suggest that SUR-associated conductances allow different neurons to respond to energy states in different ways through a common mechanism.

A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.

Background and PurposeCystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl− channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP‐628006 with a distinct chemical structure.Experimental ApproachUsing electrophysiological assays with CFTR‐expressing heterologous cells and CF patient‐derived human bronchial epithelial (hBE) cells, we compared the effects of CP‐628006 with the marketed CFTR potentiator ivacaftor.Key ResultsCP‐628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del‐CFTR were larger than those with the gating variant G551D‐CFTR. In excised inside‐out membrane patches, CP‐628006 potentiated wild‐type, F508del‐CFTR, and G551D‐CFTR by increasing the frequency and duration of channel openings. CP‐628006 increased the affinity and efficacy of F508del‐CFTR gating by ATP. In these respects, CP‐628006 behaved like ivacaftor. CP‐628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP‐628006 conferred ATP‐dependent gating on G551D‐CFTR, whereas the action of ivacaftor was ATP‐independent. For G551D‐CFTR, but not F508del‐CFTR, the action of CP‐628006 plus ivacaftor was greater than ivacaftor alone. CP‐628006 delayed, but did not prevent, the deactivation of F508del‐CFTR at the plasma membrane, whereas ivacaftor accentuated F508del‐CFTR deactivation.Conclusions and ImplicationsCP‐628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells

Insulin secretion from pancreatic β cells in response to high glucose (HG) critically depends on the inhibition of KATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of KATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6mV (from -69.2 to -38.6mV) and KATP conductance decreased by 91% (from 0.243 to 0.022nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of KATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of KATP channel surface density plays a greater role than ATP-dependent gating in regulating β cell excitability.

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

Molecular Basis for Fe(III)-Independent Curcumin Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Activity.

Curcumin potentiates the phosphorylation-dependent activity of human cystic fibrosis transmembrane conductance regulator (CFTR) in Fe(3+)-dependent and Fe(3+)-independent manners. Although the Fe(3+)-dependent curcumin potentiation results from removal of endogenous inhibitory Fe(3+) at the interface of the regulatory (R) domain and intracellular loop (ICL) 3, the molecular mechanism of Fe(3+)-independent curcumin potentiation is still unknown. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of a highly conserved ICL1/ICL4 interface and a stimulatory phosphorylation site S795 or S813 at the C-terminus of the R domain in this potentiation pathway was investigated. The results showed that highly conserved aromatic and positively charged residues at the ICL1/ICL4 interface and phosphorylation site S813 were sensitive to curcumin regardless of whether Fe(3+) and nucleotide-binding domain 2 were removed. More importantly, spontaneous disulfide cross-linking between curcumin-sensitive ICL1 and S795 was observed to be enough to promote channel opening as curcumin did. Therefore, the phosphorylated R domain, once released from ICL3, may function as a length- and gating-regulatory cross-linker between two transmembrane domains to promote the stimulatory interactions between the R domain and the ICL1/ICL4 interface. Curcumin may potentiate CFTR activity not only by removing inhibitory Fe(3+) to release the R domain from ICL3 but also by stabilizing the stimulatory R-ICL1/ICL4 interactions. Therefore, the future potentiators with two linked aromatic rings could utilize this potentiation pathway to bypass the need for ATP to rescue the activity of cystic fibrosis mutants with an ATP-dependent gating defect.

PKA-Dependent Potentiation Mechanisms of Human CFTR Activity

Both curcumin and VX-770 potentiate the channel activity of human CFTR (hCFTR) and two most common cystic fibrosis (CF) mutants G551D and F508delta in an ATP-independent but PKA-dependent manner. The underlying molecular mechanisms are unclear. Herein, HEK-293T cells cultured in a Fe3+-containing medium were transiently transfected with hCFTR constructs and curcumin with well-known chemical nature was employed as a template to explore PKA-dependent potentiation mechanisms of hCFTR activity. The results showed that curcumin potentiation of Fe3+-sensitive hCFTR activity was partially weakened by Fe3+-insensitive mutations at the interface of the R domain and intracellular loop (ICL) 3 and completely suppressed by sufficient Fe3+. Thus, release of the inhibitory Fe3+-bound R domain from ICL3 by curcumin may be critical for curcumin potentiation. Further study indicated that curcumin potentiation was significantly prohibited by a missense alanine mutation of F157, Y161 or K166 from ICL1, or R1066, F1074 or F1078 from ICL4, or S795 or S813 from the R domain with or without the involvement of nucleotide-binding domain 2 (NBD2). More importantly, curcumin potentiation was also suppressed by the R811A/S813D or Y808A/S813D mutation and disulfide crosslinking of K162C to S795C enhanced channel opening. Therefore, the phosphorylated R domain may function as a length- and gating-regulatory cross-linker between two transmembrane domains (TMD1 and TMD2). Curcumin may potentiate hCFTR activity by stabilizing the stimulatory ICL1/ICL4-R interactions that promote channel opening by pulling all ICLs together and thus triggering a gating inward-to-outward reorientation of TMDs. Possible chemical interactions may involve cation-π interactions, π-π interactions and hydrogen bonding. Taken together, both release of the R domain from ICL3 and the stimulatory R-ICL1/ICL4 interactions may be necessary for PKA-dependent hCFTR activation and potentiation. These findings may help optimize the potentiators for treating those CF mutants with an ATP-dependent gating defect.

Modulation of CFTR gating by permeant ions

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.

CFTR: a missing link between exocrine and endocrine pancreas?

CFTR: a missing link between exocrine and endocrine pancreas?

An Intramolecular Interaction Controls a Rate-Limiting Step in ATP-Dependent Gating of Kir6.2 Channels

ATP-sensitive potassium (KATP) channels are complexes of inwardly-rectifying Kir channels (Kir6.x) and sulfonylurea receptors (SUR). These two subunits work together to encode sensitivity to intracellular adenine nucleotides (ATP and ADP) and other regulatory ligands including PIP2. We investigated the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels rapidly re-open after washout of ATP, with a time constant of ∼60 ms. Extending similar kinetic measurements to mutants around the bundle crossing revealed modest effects on gating kinetics despite significant changes in ATP sensitivity. However, we identified a pair of highly conserved neighboring amino acids (Trp68, Lys170) that controls the rate of channel recovery and inhibition by ATP. Mutations of Trp68 or Lys170 have the paradoxical effect of dramatically slowing the kinetics of channel opening, while increasing channel open probability. Formalization of the transition state effects of these residues using phi-value analysis revealed a consistent steep negative slope. This finding implies that these residues play a specific role in lowering the transition state energy barrier between open and closed channel states. The demonstration of a negative phi-value is unique for an ion channel gating process, and diverges from interpretations that use phi-values to place specific residues in the spatial and temporal progression through the transition energy landscape. Using nonsense suppression for unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68, potentially required to localize the e-amine of Lys170 in the PIP2 binding site. Our findings identify a discrete pair of residues with an essential role for controlling gating kinetics of Kir channels.

Modeling the Conformational Changes Underlying Channel Opening in CFTR

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Changes in side-chain interactions are observed in all major domains of the protein, and experimental confirmation was obtained for a novel intra-protein salt bridge that breaks near the end of the transition. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.

Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels

Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the "slide helix" forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.

Converting Nonhydrolyzable Nucleotides to Strong Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Agonists by Gain of Function (GOF) Mutations

Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPγS), adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPγS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPγS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPγS removal (τdeac ∼ 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPγS activated even the strongest GOF mutant as strongly as ATP. ATPγS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPγS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.

Structure and ATP-dependent Gating Mechanism of P2X Receptors

Structure and ATP-dependent Gating Mechanism of P2X Receptors

Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single “gate” within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)2− ions was reduced when ATP concentration was reduced from 1mM to 10μM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)2− was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

Direct Interaction Of A Small Molecule Modulator With G551D-CFTR, A Cystic Fibrosis Causing Mutation Associated With Severe Disease

CFTR is a member of the ATP Binding Cassette (ABC) superfamily of membrane proteins, and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR, exhibits defective phosphorylation and ATP dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role for G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators which enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remains unclear. In the present study, we evaluated the mechanism of action of one small molecule modulator: VRT-532, identified as a specific modulator of CF causing mutants. First, we confirmed that VRT-532 caused a significant increase in channel activity by G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. This versatile assay of iodide conductance enables the study of large populations of mutant proteins in a cell-free system, removed from other confounding cellular proteins. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity by VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the wild type CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis. These studies provide rationale for using G551D as a tool for identifying the binding site of VRT-532. Studies supported by the Canadian Cystic Fibrosis Foundation and by Cystic Fibrosis Foundation Therapeutics, Inc.

Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease

CF (cystic fibrosis) is caused by mutations in CFTR (CF transmembrane conductance regulator), which cause its mistrafficking and/or dysfunction as a regulated chloride channel on the apical surface of epithelia. CFTR is a member of the ABC (ATP-binding-cassette) superfamily of membrane proteins and a disease-causing missense mutation within the ABC signature sequence; G551D-CFTR exhibits defective phosphorylation and ATP-dependent channel gating. Studies of the purified and reconstituted G551D-CFTR protein revealed that faulty gating is associated with defective ATP binding and ATPase activity, reflecting the key role of G551 in these functions. Recently, high-throughput screens of chemical libraries led to identification of modulators that enhance channel activity of G551D-CFTR. However, the molecular target(s) for these modulators and their mechanism of action remain unclear. In the present study, we evaluated the mechanism of action of one small-molecule modulator, VRT-532, identified as a specific modulator of CF-causing mutants. First, we confirmed that VRT-532 causes a significant increase in channel activity of G551D-CFTR using a novel assay of CFTR function in inside-out membrane vesicles. Biochemical studies of purified and reconstituted G551D-CFTR revealed that potentiation of the ATPase activity of VRT-532 is mediated by enhancing the affinity of the mutant for ATP. Interestingly, VRT-532 did not affect the ATPase activity of the Wt (wild-type) CFTR, supporting the idea that this compound corrects the specific molecular defect in this mutant. To summarize, these studies provide direct evidence that this compound binds to G551D-CFTR to rescue its specific defect in ATP binding and hydrolysis.

Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd2+] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd2+ is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd2+. On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Collectively, these results suggest that the effect of Cd2+ is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.

Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.