STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL.

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.

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump.

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.

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

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.

Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps.

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.

The dynamic relationships between the three events that release individual Na⁺ ions from the Na⁺/K⁺-ATPase.

Na(+)/K(+) pumps move net charge through the cell membrane by mediating unequal exchange of intracellular Na(+) and extracellular K(+). Most charge moves during transitions that release Na(+) to the cell exterior. When pumps are constrained to bind and release only Na(+), a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound Na(+), thereby transiently generating current.

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions.

Electrophysiological, biochemical, and bioinformatic methods for studying CFTR channel gating and its regulation.

CFTR is the only member of the ABC (ATP-binding cassette) protein superfamily known to function as an ion channel. Most other ABC proteins are ATP-driven transporters, in which a cycle of ATP binding and hydrolysis, at intracellular nucleotide binding domains (NBDs), powers uphill substrate translocation across the membrane. In CFTR, this same ATP-driven cycle opens and closes a transmembrane pore through which chloride ions flow rapidly down their electrochemical gradient.

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation.

Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations.

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.

Visualizing the mapped ion pathway through the Na,K-ATPase pump.

The Na(+),K(+)-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na(+) ions for extracellular K(+) ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel.

Ion channels versus ion pumps: the principal difference, in principle.

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.

Review. Peering into an ATPase ion pump with single-channel recordings.

In principle, an ion channel needs no more than a single gate, but a pump requires at least two gates that open and close alternately to allow ion access from only one side of the membrane at a time. In the Na+,K+-ATPase pump, this alternating gating effects outward transport of three Na+ ions and inward transport of two K+ ions, for each ATP hydrolysed, up to a hundred times per second, generating a measurable current if assayed in millions of pumps. Under these assay conditions, voltage jumps elicit brief charge movements, consistent with displacement of ions along the ion pathway while one gate is open but the other closed.

The ion pathway through the opened Na(+),K(+)-ATPase pump.

P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane to avoid the dissipation of the gradients that would occur during simultaneous access. In Na(+),K(+)-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3).

Sodium flux ratio in Na/K pump-channels opened by palytoxin.

Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells.

Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.

CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2).

In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer.

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 NH(2)-terminal NBD1 and another in the COOH-terminal NBD2.

Ion permeation through the Na+,K+-ATPase.

P-type ATPase pumps generate concentration gradients of cations across membranes in nearly all cells. They provide a polar transmembrane pathway, to which access is strictly controlled by coupled gates that are constrained to open alternately, thereby enabling thermodynamically uphill ion transport (for example, see ref. 1).

Ouabain affinity determining residues lie close to the Na/K pump ion pathway.

The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain.

The ABC protein turned chloride channel whose failure causes cystic fibrosis.

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.

Control of the CFTR channel's gates.

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.

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.

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).

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR.