Phosphorylation and activation of a bovine tracheal anion channel by Ca2+/calmodulin-dependent protein kinase II

Academic Article

Abstract

  • Secretion of Cl by epithelial cells is fundamental to the processes of fluid and electrolyte transport by epithelia such as those of the airways, sweat-ducts, and gastrointestinal tract. In the present study, we show that a novel Cl channel protein, immunoaffinity purified from bovine tracheal apical membrane vesicles, is sensitive to phosphorylation by Ca /calmodulin protein kinase II (CaMK II). The channel protein, which migrates with an M(r) of 140,000 under nonreducing conditions, is phosphorylated in vitro by CaMK II in a Ca - and calmodulin-dependent manner. When reconstituted into planar lipid bilayers, the protein behaves as an anion-selective, 4,4'-diisothiocyanostilbene- and dithiothreitol-sensitive channel. The open probability of this channel is significantly increased by Ca alone but only at levels of Ca (5-10 μM) that lie outside the physiological range. Addition of CaMK II to the presumptive cytoplasmic side of the bilayer in the presence of ATP and calmodulin dramatically increased the sensitivity of the channel to free Ca , shifting the dose-response curve for Ca -dependent channel activation to lower [Ca ](i), the maximum increase in channel P(o) occurring between 0.6 and 1 μM. The addition of kinase in the absence of ATP or calmodulin or the addition of ATP or calmodulin in the absence of kinase was without effect on channel P(o). Increasing [Ca ] above 1 μM decreased channel mean current, causing a flickery block that was maximal at 2 μM. Increasing [Ca ] as high as 10 μM in the presence of kinase did not further alter channel behavior. In contrast to CaMK II, the addition of the catalytic subunit of protein kinase A either alone or together with ATP had no effect on channel P(o). These observations suggest that a novel Ca -sensitive anion channel isolated from bovine airway epithelium is regulated by CaMK II phosphorylation. - - 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+
  • Published In

    Author List

  • Fuller CM; Ismailov II; Keeton DA; Benos DJ
  • Start Page

  • 26642
  • End Page

  • 26649
  • Volume

  • 269
  • Issue

  • 43