ACTIVATION OF EPITHELIAL NA-A REQUIRES ACTIN-FILAMENTS( CHANNELS BY PROTEIN KINASE)

We have recently demonstrated a novel role for ''short'' actin filamen ts, a distinct species of polymerized actin different from either mono meric (G-actin) or long actin filaments (F-actin), in the activation o f epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic mo nophosphate-dependent protein kinase A (PKA) was investigated by patch -clamp techniques in A6 epithelial cells. In excised inside-out patche s, addition of deoxyribonuclease I, which prevents actin polymerizatio n, inhibited Na+ channel activation mediated by PKA. Disruption of end ogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activ ation following the addition of actin to the cytosolic side of the pat ch. To assess the role of PKA on actin filament organization, actin wa s used as a substrate for the specific phosphorylation by the PKA. Act in was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin r educed the rate of actin polymerization. Thus actin allowed to polymer ize for at least 1 h in the presence of PKA and ATP to obtain phosphor ylated actin filaments induced Na+ channel activity in excised inside- out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphory lated G-actin incubated in a polymerizing buffer for at least 1 h at 3 7-degrees-C. These data suggest that the form of actin required for Na + channel activation (i.e., ''short'' actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate t he activation of Na+ channels by PKA.