The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport ...across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
The incessant traffic of ions across cell membranes is controlled by two kinds of border guards: ion channels and ion pumps. Open channels let selected ions diffuse rapidly down electrical and ...concentration gradients, whereas ion pumps labour tirelessly to maintain the gradients by consuming energy to slowly move ions thermodynamically uphill. Because of the diametrically opposed tasks and the divergent speeds of channels and pumps, they have traditionally been viewed as completely different entities, as alike as chalk and cheese. But new structural and mechanistic information about both of these classes of molecular machines challenges this comfortable separation and forces its re-evaluation.
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of ...dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR’s channel function.
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•Molecular structure of human CFTR in the dephosphorylated, ATP-free form•Correlation between CFTR channel gating cycle and ATP hydrolysis rates•Identification of a structure feature distinguishing CFTR from other ABC transporters
Molecular structure of human CFTR determined in the dephosphorylated, ATP-free conformation provides insights on the structural basis for its channel activity.
CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which ...in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.
A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump ...autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.
Mutations in the neuron-specific α3 isoform of the Na+/K+-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related ...movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α3+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron in vivo recordings in awake α3+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na+/K+ exchange, but allowed the pumps to bind Na+ and become phosphorylated. These findings implicate aberrant cerebellar activity in α3 isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α3 isoform Na+/K+-ATPase.
CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic ...nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.
ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the ...protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.
The human ATP‐binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight ...into the dynamic interaction between CFTR's nucleotide‐binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl‐specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH2‐terminal NBD1 and another in the COOH‐terminal NBD2. Identification of crosslinked product was facilitated by co‐expression of NH2‐terminal and COOH‐terminal CFTR half channels each containing one NBD. The COOH‐terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type‐like, phosphorylation‐ and ATP‐dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head‐to‐tail configuration analogous to that in homodimeric crystal structures of nucleotide‐bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head‐to‐tail NBD1–NBD2 association to channel opening.
The Na+/K+pump is a ubiquitous P-type ATPase that binds three cytoplasmic Na+ions deep within its core where they are temporarily occluded before being released to the extracellular surface. The ...3Na+/2K+-exchange transport cycle is completed when two extracellular K+ions bind and become temporarily occluded within the protein and subsequently released to the cytoplasm. Coupling of Na+-ion occlusion to phosphorylation of the pump by ATP and of K+-ion occlusion to its dephosphorylation ensure the vectorial nature of net transport. The occluded-ion conformations, with binding sites inaccessible from either side, represent intermediate states in these alternating-access descriptions of transport. They afford protection against potentially catastrophic effects of inadvertently allowing simultaneous access from both membrane sides. The marine toxin, palytoxin, converts Na+/K+pumps into nonselective cation channels, possibly by disrupting the normal strict coupling between opening of one access pathway in the Na+/K+ATPase and closing of the other. We show here that gating of the channels in palytoxin-bound Na+/K+pumps in excised membrane patches is modulated by the pump's physiological ligands: cytoplasmic application of ATP promotes opening of the channels, and extracellular replacement of Na+ions by K+ions promotes closing of the channels. This suggests that, despite the presence of bound palytoxin, certain partial reactions of the normal Na+/K+-transport cycle persist and remain capable of effecting the conformational changes that control access to the pump's cation-binding sites. These findings affirm the alternating-access model of ion pumps and offer the possibility of examining ion occlusion/deocclusion reactions in single pump molecules.
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