Blocking action of cytochalasin D on protein kinase A stimulation of a stretch-activated cation channel in renal epithelial A6 cells

Abstract

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.