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.
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.
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.ATPase cycle | maximum likelihood | nonequilibrium | phosphorylation | Walker motifsA TP-binding cassette (ABC) proteins bind and hydrolyze ATP, usually to power transport of substrates across membranes. Their common architecture comprises two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs) containing conserved sequences for interacting with ATP. Once ATP binds to the conserved Walker A and B motifs of each NBD, they dimerize in head-to-tail fashion, burying two ATP molecules in composite interfacial sites (1, 2). Cyclic formation and disruption of the dimer requires hydrolysis of at least one of the ATPs per cycle (3-6).The ion channel CFTR contains, in addition to canonical ABC protein domains (TMD1, NBD1, TMD2, NBD2), a unique regulatory (R) domain (7) with multiple cAMP-dependent protein kinase (PKA) targets that must be phosphorylated for ATP to activate bursts of channel openings reviewed in ref. 8). But the mechanism of CFTR channel gating remains controversial. Early observations that the gating pattern violates microscopic reversibility suggested that the process underlying bursting operates far from thermodynamic equilibrium (9-11). Building on this, with functional evidence from mutants and nucleotide homologs (12-15) and with structural and biochemical data from prokaryotic NBDs (1-4), a CFTR channel open burst was proposed to be initiated by ATP-mediated formation of a stable NBD1-NBD2 heterodimer, and terminated by dimer dissociation after ATP hydrolysis (16). The ATP hydrolysis was posited to occur at the NBD2 composite site (between NBD2 Walke...
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.
Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the ~3 Å resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post-filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.
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.
A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.
TRPM2 is a tetrameric Ca2+-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding sites for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca2+ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ∼50-pS nonselective cation channels; K1/2 for ADPR was ∼1 µM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1 mM of free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca2+ likely triggers prolonged, self-sustained TRPM2 activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.