State of ClCa phosphorylation affects the pharmacology of niflumic acid in rabbit pulmonary artery smooth muscle cells

Abstract

Ca2+ dependent Cl− currents (IClCa) are important modulators of membrane excitability in vascular smooth muscle cells. The underlying channel (ClCa) appears to be negatively regulated by CaMKII‐induced phosphorylation, and up regulated by the phosphatase calcineurin. Niflumic acid (NFA), considered the most potent blocker of IClCa in many cell types, both blocks and paradoxically enhances IClCa in pulmonary artery (PA) myocytes. This study aimed to determine if the state of channel phosphorylation affects the pharmacology of NFA in dispersed rabbit PA myocytes. IClCa was evoked by 500 nM free Ca2+. The ability of NFA to inhibit IClCa was greatly reduced in conditions promoting phosphorylation. NFA also appeared to have a dual effect on IClCa when applied at low concentrations (0.1‐10 μM), enhancing IClCa in the presence of 5 mM ATP, while blocking IClCa in cells dialyzed with10 µM KN‐93 (CaMKII inhibitor) and no ATP. Analysis of the voltage‐dependence of interaction of NFA with IClCa showed that while the maximal level of block at positive potentials and stimulation at negative potentials were similar, dephosphorylation shifted the voltage at which conversion from block to stimulation occurs by more than ‐80 mV. These results show that the state of phosphorylation has a profound influence on the interaction of NFA with ClCa channels, which may explain the wide range of sensitivities to NFA reported in many studies.