The Interaction of Antrhacene-9-Carboxylic Acid with Calcium-Activated Chloride Channels is Influenced by the State of Global Phosphorylation in Pulmonarty Artery Smooth Muscle Cells

Abstract

Ca2+-dependent Cl- currents (IClCa) are inhibited by phosphorylation in arterial smooth muscle cells. We recently reported that niflumic acid (NFA), an inhibitor of IClCa, is less efficacious at blocking these currents in conditions promoting phosphorylation (Wiwchar et al., Br J Pharmacol, in press, 2009). This study aimed to assess whether another Cl- channel blocker, anthracene-9-carboxylic acid (A9C), is also affected by channel phosphorylation. A9C blocks IClCa at positive potentials but paradoxically stimulates the inward IClCa tail after repolarization to negative potentials (Piper & Greenwood, Br J Pharmacol 138: 31-38, 2003). IClCa was evoked by pipette solutions containing 500 nM free Ca2+ with or without 5 mM ATP to alter the state of phosphorylation. Although A9C (1-500 uM) dose-dependently blocked steady state IClCa at potentials positive to 0 mV in all cell groups, its maximal effect and sensitivity to voltage were enhanced in cells dialyzed with 0 vs. 5 mM ATP. For example, maximal block by 100 uM A9C was 35 and 73%, and V0.5 was 110 and 67 mV, in cells with 5 vs 0 ATP, respectively. A9C enhanced IClCa tail at −80 mV by causing a negative shift in voltage-dependence in both cell groups, with a larger shift occurring in cells dialyzed with 5 mM ATP. Interestingly, 100 but not 500 uM A9C stimulated steady-state ICl(Ca) at potentials < 0 mV in cells dialyzed with 0 ATP, a potential range where ICl(Ca) was unaffected in myocytes dialyzed with 5 mM ATP. As with NFA, the complex actions of A9C on IClCa are influenced by the state of channel phosphorylation and we propose the existence of at least two binding sites with different affinities for A9C.