Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl^- secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal LIGHT immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl^- secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl^- secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (I_sc, equal to Cl^- secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.