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) Fe(3+) 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 Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) 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 Fe(3+) 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 Fe(3+) 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 Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.
Department of Physiology and Biophysics and the Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA. wangg@medicine.nevada.edu
Guangyu
G
Wang
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002614:Chelating Agents / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007246:Infertility / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007501:Iron / Q000378:metabolism*; D020125:Mutation, Missense; D010618:Phenanthrolines / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
C505032:CFTR protein, human; D002614:Chelating Agents; D010618:Phenanthrolines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D007501:Iron