Background: In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.

Epithelial sodium channels (ENaCs) are located on the apical surface of cells and funnel Na(+) ions from the lumen into the cell. ENaC function also regulates extracellular fluid volume as water flows across membranes accompanying Na(+) ions to maintain osmolarity. To examine the sites of expression and intracellular localization of ENaC, we generated polyclonal antibodies against the extracellular domain of human α-ENaC subunit that we expressed in E. coli. Three-dimensional (3D) confocal microscopy of immunofluorescence using these antibodies for the first time revealed that ENaCs are uniformly distributed on the ciliary surface in all epithelial cells with motile cilia lining the bronchus in human lung and female reproductive tract, all along the fimbrial end of the fallopian tube, the ampulla and rare cells in the uterine glands. Quantitative analysis indicated that cilia increase cell surface area >70-fold and the amount of ENaC on cilia is >1,000-fold higher than on non-ciliated cell surface. These findings indicate that ENaC functions as a regulator of the osmolarity of the periciliary fluid bathing the cilia. In contrast to ENaC, cystic fibrosis transmembrane conductance regulator (CFTR) that channels chloride ions from the cytoplasm to the lumen is located mainly on the apical side, but not on cilia. The cilial localization of ENaC requires reevaluation of the mechanisms of action of CFTR and other modulators of ENaC function. ENaC on motile cilia should be essential for diverse functions of motile cilia, such as germ cell transport, fertilization, implantation, clearance of respiratory airways and cell migration.

Severe PHA cases are associated with mutations leading to absence of normal-length alpha, beta or gammaENaC, while a mild case has been found to be associated with a missense mutation in alphaENaC. The predominance of PHA-causing mutations in the alphaENaC gene may be related to the function of this subunit.