State-dependent Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gating by a High Affinity Fe3+ Bridge between the Regulatory Domain and Cytoplasmic Loop 3*

The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 × 10−19 m) Fe3+ bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe3+ binding. More importantly, inhibition by Fe3+ was state-dependent. Sensitivity to Fe3+ was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe3+ no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe3+ may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe3+ homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

[1]  Jianping Wu,et al.  ATP-independent CFTR channel gating and allosteric modulation by phosphorylation , 2010, Proceedings of the National Academy of Sciences.

[2]  J. Riordan,et al.  Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding. , 2009, Journal of structural biology.

[3]  A. Mehta,et al.  Mechanistic Insight into Control of CFTR by AMPK*S⃞ , 2009, Journal of Biological Chemistry.

[4]  D. Clarke,et al.  Processing Mutations Disrupt Interactions between the Nucleotide Binding and Transmembrane Domains of P-glycoprotein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)* , 2008, Journal of Biological Chemistry.

[5]  Nikolay V. Dokholyan,et al.  Multiple Membrane-Cytoplasmic Domain Contacts in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mediate Regulation of Channel Gating* , 2008, Journal of Biological Chemistry.

[6]  K. Locher,et al.  Structural Basis of Trans-Inhibition in a Molybdate/Tungstate ABC Transporter , 2008, Science.

[7]  Adrian W. R. Serohijos,et al.  Phenylalanine-508 mediates a cytoplasmic–membrane domain contact in the CFTR 3D structure crucial to assembly and channel function , 2008, Proceedings of the National Academy of Sciences.

[8]  Geoffrey Chang,et al.  Flexibility in the ABC transporter MsbA: Alternating access with a twist , 2007, Proceedings of the National Academy of Sciences.

[9]  Ying Wang,et al.  Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein* , 2007, Journal of Biological Chemistry.

[10]  J. Forman-Kay,et al.  CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices , 2007, Nature Structural &Molecular Biology.

[11]  M. Covarrubias,et al.  Zn2+-dependent Redox Switch in the Intracellular T1-T1 Interface of a Kv Channel* , 2007, Journal of Biological Chemistry.

[12]  Y. Sohma,et al.  G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects , 2007, The Journal of general physiology.

[13]  K. Kirk,et al.  Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains* , 2007, Journal of Biological Chemistry.

[14]  Andrei Aleksandrov,et al.  Domain interdependence in the biosynthetic assembly of CFTR. , 2007, Journal of molecular biology.

[15]  Angus C Nairn,et al.  In vivo phosphorylation of CFTR promotes formation of a nucleotide‐binding domain heterodimer , 2006, The EMBO journal.

[16]  R. Dawson,et al.  Structure of a bacterial multidrug ABC transporter , 2006, Nature.

[17]  A. Nairn,et al.  Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA , 2005, The Journal of general physiology.

[18]  H. Hellinga,et al.  Orthogonal site‐specific protein modification by engineering reversible thiol protection mechanisms , 2005, Protein science : a publication of the Protein Society.

[19]  Rugang Zhang,et al.  Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation. , 2004, American journal of physiology. Cell physiology.

[20]  G. Yellen,et al.  Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges , 2004, Nature.

[21]  J. M. Sauder,et al.  Structure of nucleotide‐binding domain 1 of the cystic fibrosis transmembrane conductance regulator , 2004, The EMBO journal.

[22]  P. Aisen,et al.  The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  John F Hunt,et al.  ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. , 2002, Molecular cell.

[24]  Junxia Xie,et al.  A Short Segment of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Contains Channel Stimulatory and Inhibitory Activities That Are Separable by Sequence Modification* , 2002, The Journal of Biological Chemistry.

[25]  D. McRee,et al.  Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein. , 2001, Biochemistry.

[26]  Joseph F. Cotten,et al.  Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels with R Domain Deletions and Translocations Show Phosphorylation-dependent and -independent Activity* , 2001, The Journal of Biological Chemistry.

[27]  M. Welsh,et al.  Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  A. D. Robertson,et al.  A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  B. Nilius,et al.  Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs. , 1999, Biochemistry.

[30]  J. Blalock,et al.  CFTR chloride channel regulation by an interdomain interaction. , 1999, Science.

[31]  Joseph F. Cotten,et al.  Covalent Modification of the Regulatory Domain Irreversibly Stimulates Cystic Fibrosis Transmembrane Conductance Regulator* , 1997, The Journal of Biological Chemistry.

[32]  J. Riordan,et al.  Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. , 1994, Biochemistry.

[33]  J. Riordan,et al.  Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR) , 1992, Cell.

[34]  M. Welsh,et al.  Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel , 1991, Cell.

[35]  L. Tsui,et al.  Erratum: Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA , 1989, Science.

[36]  H. Birkedal‐Hansen,et al.  Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation. , 1990, Proceedings of the National Academy of Sciences of the United States of America.