Blocking action of cytochalasin D on protein kinase A stimulation of a stretch-activated cation channel in renal epithelial A6 cells.
We have shown that the apical membrane of renal epithelial A6 cells has a 29-pS stretch-activated nonselective cation (NSC) channel, which is activated by cytosolic cyclic AMP (cAMP) (J Gen Physiol 1997;110:327-36). In general, downstream signalings of cAMP are mediated through a cAMP-activated protein kinase (protein kinase A, PKA)-dependent pathway. Therefore, to study if the channel is activated by a PKA-dependent pathway, we applied a PKA catalytic subunit directly to the channel from the cytosolic surface in cytosol-free excised inside-out patches, using the single channel recording (patch clamp) technique. Application of PKA catalytic subunit with 2 mM ATP increased the open probability (P(o)) of the channel from 0.11 +/- 0.04 to 0.58 +/- 0.10 (mean +/- SD, N = 11, P < 0.001). The channel has a gating kinetics "C(L) <--> C(S) <--> O, " where C(L,) C(S,) and O are the long closed state, the short closed state, and the open state, respectively. PKA influenced the communication of the channel between C(L) and C(S) without affecting the communication between C(S) and O, leading the channel to only stay in C(S) and O. The PKA-induced increase in P(o) was attributable to the interruption of communication between C(L) and C(S) or to the reduction of time the channel stays in C(L.) Pretreatment with cytochalasin D (Cyt-D), an inhibitor of the polymerization of actin filaments, blocked the stimulatory effect of PKA on the channel. These observations suggest that phosphorylation of polymerized actin filaments regulates the gating kinetics of a stretch-activated channel in renal epithelium.
Department of Cellular and Molecular Physiology, Kyoto Prefectural University of Medicine, 602-0841, Japan.
N
N
Niisato
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Y
Marunaka
D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D014982:Xenopus laevis
D007473:Ion Channels; D019384:Nucleic Acid Synthesis Inhibitors; D015638:Cytochalasin D; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases