Activation of epithelial Na+ channels by protein kinase A requires actin filaments.
We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin 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 reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated 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 phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 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 the activation of Na+ channels by PKA.
Prat
A G
AG
Renal Unit, Massachusetts General Hospital, Charlestown 02129.
Bertorello
A M
AM
Ausiello
D A
DA
Cantiello
H F
HF
D000199:Actins / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004594:Electrophysiology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011494:Protein Kinases / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology
D000199:Actins; D011108:Polymers; D015222:Sodium Channels; D015638:Cytochalasin D; D011494:Protein Kinases; D003850:Deoxyribonuclease I