The Role of the R-domain in Regulated Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator.

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Christopher Michael Lewarchik
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated chloride channel that is a member of the ATP-binding cassette (ABC) transporter family [1]. It is involved in the movement of chloride ions across epithelial membranes in the airways, sweat glands, intestine and pancreas [2]. Mutations in CFTR that result in a loss of channel function result in the disease cystic fibrosis, affecting nearly 1 in 2500 people in northern Europe and the United States [3]. As a member of the ABC transporter family, CFTR shares the structural features of these proteins. Unique to CFTR is the presence of a cytoplasmic R-domain, that contains multiple phosphorylation sites. Phosphorylation of the R domain is required for CFTR channel gating, and cAMP/PKA simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments [4]. We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R-domain (ƒ´aa 635-834) produced a channel with elevated basal current (Im) and no ƒ´Im or trafficking response (ƒ´Cm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838 produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. This 22aa region exhibits helical structure, bears a net negative charge of -9, is highly conserved among species, and has been termed NEG2 [5, 6]. Injection of NEG2 peptide into oocytes expressing split ƒ´NEG2 CFTR prior to stimulation restored the agonist-evoked ƒ´Cm, consistent with the concept that this sequence mediates regulated CFTR trafficking. Further modifications of NEG2 suggest that the trafficking phenotype depends primarily on its helical structure. These observations suggest that the NEG2 region at the C-terminus of the R domain allows CFTR to enter a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA-stimulation.