Phosphorylation alters the pharmacology of Ca2+-activated Cl- channels in rabbit pulmonary arterial smooth muscle cells

Background and purpose: Ca2+-activated Cl- currents (I-Cl(Ca)) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca2+-activated Cl- channel (Cl-Ca) blocker niflumic acid (NFA) produces a paradoxical dual effect on I-Cl(Ca), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I-Cl(Ca) were modulated by phosphorylation. Experimental approach: I-Cl(Ca) was elicited with 500 nM free internal Ca2+ in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 mu M). Key results: Dephosphorylation enhanced the ability of 100 mu M NFA to inhibit I-Cl(Ca). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, similar to 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I-Cl(Ca) in the range of 0.1-250 mu M in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 mu M, which converted to block at concentrations > 1 mu M when cells were dialysed with 5 mM ATP. Conclusions and implications: Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl-Ca channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.