title: Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism.
abstract: Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.
authors.affiliation: Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Saxena
authors.forename: Simarna
authors.initials: S
authors.lastname: Kaur
mesh_terms: D002352:Carrier Proteins / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology; D020691:rab GTP-Binding Proteins / Q000502:physiology*; D000075745:rab27 GTP-Binding Proteins
chemical_list: C505032:CFTR protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; C457772:SYTL5 protein, human; C507969:UNC13D protein, human; D000075745:rab27 GTP-Binding Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins