LARSEN, E. H.*; AMSTRUP, J.; WILLUMSEN, N.J.; University of Copenhagen, Denmark: Function, regulation and cloning of CFTR in toad (Bufo bufo) skin mitochondria rich cells

Amphibian skin absorbs NaCl and H₂O by epidermal cells and secretes fluid by subepidermal glands. CFTR is implicated in both functions. The epidermis contains Na transporting principal cells and Cl transporting mitochondria-rich cells (MRC). MRC-Cl conductance is activated by voltage when the electrical gradient favors uptake of Cl. An anion conductance is activated also by the β-adrenergic agonist isoproterenol, G_Cl > G_Br > G_NO3 > G_I > G_gluconate = 0. Post isoproterenol the Cl conductance is further increased by genistein and reduced by glibenclamide. Monoclonal antibodies (hCFTR aa 1377-1480 of C-terminus and 590-830 of R-domain) revealed selective expression of CFTR in MRC. The apical membrane of MRC harbors a forskolin activated GHK-rectifying Cl channel with conductance, γ_125/125 = 8 pS. PCR with mRNA from gland free epithelium resulted in specific amplification of a targeted 640 bp region of xCFTR-cDNA. Cloned full-length toad bbCFTR has several regions of homology with hCFTR with an overall identity of 89% and highly conserved regions corresponding to the R-domain and the Walker A and B motifs of NBD1 and NBD2 (GenBank #AYO26761). Unlike hCFTR, but similar to lower vertebrate CFTR, bbCFTR contains a PKC consensus site at Thr⁶⁶³. Receptor stimulation results in rightward displacement of the G_Cl-V relationship with no increase of the fully V-activated conductance, i.e., the two ways of fully activating the MRC-Cl conductance are mutually exclusive. Noise analysis of V-activated whole-cell MRC-Cl currents indicated activity of a 250-pS channel and thus a channel protein different from CFTR.