Regulation of CFTR ion channel gating by MgATP

AbstractSingle channel currents of wild-type CFTR reconstituted in lipid bilayers were recorded to study the temperature dependence of channel gating between +20°C and +40°C. The opening of the channel was highly temperature dependent and required an activation energy of about 100 kJ/mol. Closing of the channel was only weakly temperature dependent with an activation energy close to that of diffusion in water. We found no significant difference in the free energy between the open and closed states. Most of the excess energy needed to activate channel opening is used to diminish the entropy of the open state. This structural reorganization is initiated by ATP binding followed by interconversion to the open channel structure as the CFTR-ATP-Mg complex passes to the transition state for hydrolysis. The energy of the CFTR-ATP-Mg interaction in the transition state is responsible for the CFTR ion channel opening rather than the energy of ATP hydrolysis. Channel closing is a diffusion limited process and does not require additional ATP binding

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR’s mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50°C and remain monodisperse at 4°C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed

Regulatory Insertion Removal Restores Maturation, Stability and Function of ΔF508 CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multi-domain membrane protein which matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32 residue segment termed the regulatory insertion (RI) not present in other ABC transporters. We report here that RI deletion enabled ΔF508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14h in cells, similar to the wild-type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermo-stabilizing influence on the channel with gating up to at least 40°C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508 containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation

Rational coupled dynamics network manipulation rescues disease-relevant mutant cystic fibrosis transmembrane conductance regulator

A novel approach identifying networks of residues involved in trans -protein dynamic coupling is applied to rescue mutant CFTR

Allosteric Modulation Balances Thermodynamic Stability and Restores Function of ΔF508 CFTR

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state

Restoration of NBD1 Thermal Stability Is Necessary and Sufficient to Correct ∆F508 CFTR Folding and Assembly

CFTR (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis (CF). The most common CF-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question we varied the extent of stabilization of NBD1 using different second site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ΔF508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function

Complex Curve of the Two Matrix Model and its Tau-function

We study the hermitean and normal two matrix models in planar approximation for an arbitrary number of eigenvalue supports. Its planar graph interpretation is given. The study reveals a general structure of the underlying analytic complex curve, different from the hyperelliptic curve of the one matrix model. The matrix model quantities are expressed through the periods of meromorphic generating differential on this curve and the partition function of the multiple support solution, as a function of filling numbers and coefficients of the matrix potential, is shown to be the quasiclassical tau-function. The relation to softly broken N=1 supersymmetric Yang-Mills theories is discussed. A general class of solvable multimatrix models with tree-like interactions is considered.Comment: 36 pages, 10 figures, TeX; final version appeared in special issue of J.Phys. A on Random Matrix Theor

History effects and pinning regimes in solid vortex matter

We propose a phenomenological model that accounts for the history effects observed in ac susceptibility measurements in YBa2Cu3O7 single crystals [Phys. Rev. Lett. 84, 4200 (2000) and Phys. Rev. Lett. 86, 504 (2001)]. Central to the model is the assumption that the penetrating ac magnetic field modifies the vortex lattice mobility, trapping different robust dynamical states in different regions of the sample. We discuss in detail on the response of the superconductor to an ac magnetic field when the vortex lattice mobility is not uniform inside the sample. We begin with an analytical description for a simple geometry (slab) and then we perform numerical calculations for a strip in a transverse magnetic field which include relaxation effects. In calculations, the vortex system is assumed to coexist in different pinning regimes. The vortex behavior in the regions where the induced current density j has been always below a given threshold (j_c^>) is described by an elastic Campbell-like regime (or a critical state regime with local high critical current density, j_c^>). When the VS is shaken by symmetrical (e.g. sinusoidal) ac fields, the critical current density is modified to j_c^) at regions where vortices have been forced to oscillate by a current density larger than j_c^>. Experimentally, an initial state with high critical current density (j_c^>) can be obtained by zero field cooling, field cooling (with no applied ac field) or by shaking the vortex lattice with an asymmetrical (e.g. sawtooth) field. We compare our calculations with experimental ac susceptibility results in YBa2Cu3O7 single crystals.Comment: 11 pages, 7 figures. To be published in PR