Phosphorylation alters the pharmacology of Ca(2+)-activated Cl channels in rabbit pulmonary arterial smooth muscle cells.
OBJECTIVE
Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-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.


METHODS
I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) 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 microM).


RESULTS
Dephosphorylation enhanced the ability of 100 microM 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, approximately 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 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP.


CONCLUSIONS
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.
Department of Pharmacology, Center of Biomedical Research Excellence (COBRE), University of Nevada School of Medicine, Reno, NV 89557-0270, USA.
M
M
Wiwchar
R
R
Ayon
I A
IA
Greenwood
N
N
Leblanc
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors; D018118:Chloride Channels / Q000378:metabolism*; D066298:In Vitro Techniques; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009544:Niflumic Acid / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
D000255:Adenosine Triphosphate; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D018118:Chloride Channels; D009544:Niflumic Acid; D021122:Protein Subunits; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides