SHRINKAGE ACTIVATES A NONSELECTIVE CONDUCTANCE - INVOLVEMENT OF A WALKER-MOTIF PROTEIN AND PKC

The ability of all cells to maintain their volume during an osmotic ch allenge is dependent on the regulated movement of salt and water acros s the plasma membrane. We demonstrate the phosphorylation-dependent ga ting of a nonselective conductance in Caco-2 cells during cellular shr inkage. Intracellular application of exogenous purified rat brain prot ein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na+-to-Cl- permeability ratio o f similar to 1.7:1. To prevent possible PKC- and/or shrinkage-dependen t activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR(505-511), was introduced into the cells via the p atch pipette. Anti-CFTR(505-511), which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC /shrinkage-induced current activation. The peptide CFTR(505-511) also induced current inhibition, suggesting the possible involvement of a r egulatory element in close proximity to the channel that shares sequen ce homology with the first nucleotide binding fold of CFTR and whose b inding to the channel is required for channel gating.