BACK TO DIAGRAM CFTR REVIEW PAGE
EXTRACELLULAR LOOP 1
GRIIASYDPDNKEER
(amino acids 103 to 117)
The extracellular loops of ion channels reside in the outside environment of the cell and usually consist of amino acids that are hydrophilic in nature, such as arginine [R], serine [S], and glutamic acid [E]. Some studies suggest ecl1 may be involved in forming part of the pore, and the external vestibule of the pore may be formed in part by ECL1. CFTR itself is predicted to be 77% present in the cytoplasm of the cell (intracellular loops, NBDs, R-domain), 4% of its total as extracellular loops, and 19% in membrane-spanning segments. The extracellular loops of CFTR are very short compared to the intracellular loops, except possibly ECL1 and ECL4, and when comparing this loop to the same loop in CFTR from other species, it is seen that there is divergence in the sequence.
RESEARCH RESULTS
Structural prediction algorithms were used to predict that this loop is primarily unstructured and irregular. It has been shown by engineering glycosylation sites into all predicted loops, both intracellular as well as extracellular that the structure originally predicted for CFTR is so far correct as to the placement of the loops.
In the MsbA crystal structure, ECL1 is from residues 53-64, and was found to cross over to the opposite side of the protein (i.e. the TM helices are not in order in either monomer, but rather go 2, 3, 4, 1, 6). The electron densities for residues 58-63 of MsbA were disordered. Science 9/7/01, Vol 293 pgs 1793-1800
The FLAG epitope has been engineered into ECL1 and the protein was shown to be functional and correctly targeted to the apical membrane.
R117C/H/L/P are known CF-causing mutations, suggesting this arginine is important in this loop. It has also been suggested that the arginine is involved in sensing extracellular pH and thereby influencing conduction. Gating is the most effected property rather than conductance by the R117H mutation and this mutation results in a channel open probability 1/3 that of wild-type. Both single-channel conductance and the open probability of the pore are affected, but not anion selectivity. The R117H mutation results in a milder phenotype of CF than the deltaF508. It reduces chloride conductance by ~70%. It has been suggested that changes in this loop affect gating (which has been shown to take place in the cytoplasm) due probably to allosteric as opposed to direct effects. Residues in this loop may have a different role than the intracellular loops, which are apparently involved in gating and stabilization of conductance states.
A series of truncation mutants were constructed in 1995 and it was found that CFTR missing the first 118 amino acids functioned similarly to wild-type, but had a smaller conductance and open probability. Mutants without transmembrane helix 1 can therefore form active chloride channels.
Residues in extracellular loop 1 have been shown to influence the selectivity of the channel to anions. For example, changing 3 amino acids to produce the same loop as CFTR in Xenopus produces the same anion selectivity.
Binding occurs between the outer core of the bacterial lipopolysaccharide of Pseudomonas and amino acids 108-117 of CFTR in extracellular loop 1 of CFTR and may be how epithelial cells are able to internalize and rid this bacterium from the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Immunoelectron microscopy revealed that more CFTR bound S. typhi in the submucosa of Cftr wildtype mice than in delta-F508 heterozygous mice. Proc Natl Acad Sci U S A 2000 Aug 1;97(16):8822-8
In May, 2001, Hammerle et al. reported on a study involving
extracellular loop contributions to both maturation and chloride
activity. They stated that "The first and fourth
extracytoplasmic loops (ELs) contain approximately 15 and 30 residues,
respectively; the other four ELs are extremely short. To examine
the influence of missense mutants in ELs detected in patients with cystic
fibrosis, we have expressed them in mammalian (baby hamster kidney (BHK21))
cells and assessed their biosynthetic processing and chloride channel activity.
In contrast to previous findings that 18 of 30 disease-associated
missense mutations in cytoplasmic loops caused retention of the nascent
polypeptides in the endoplasmic reticulum, all the EL mutants studied matured
and were transported to the cell surface. This pronounced asymmetry
is consistent with the notion that endoplasmic reticulum quality control of
nascent CFTR is exerted primarily on the cytoplasmic side of the membrane.
Although this set of EL mutations has little effect on CFTR maturation, most of
them seriously compromise its chloride channel activity. Substitutions at six
different positions in EL1 and single positions in EL2 and EL4 all destabilized
the open state, some of them severely, indicating that the ELs contribute to the
stability of the CFTR ion pore." J Biol Chem 2001 May
4;276(18):14848-54