Modulation of Ca2+‐dependent Cl− channels by calcineurin in rabbit coronary arterial myocytes

Abstract

The role of the Ca²⁺‐dependent phosphatase calcineurin (CaN) in the modulation of Ca²⁺‐dependent Cl^‐ channels (Cl_Ca) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin‐dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca²⁺ was increased. Cl_Ca currents (I_Cl(Ca)) elicited by cell dialysis with fixed intracellular Ca²⁺ levels up to 500 nm were inhibited by 10 μm cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage‐dependent manner, whereas currents evoked by 1 μm Ca²⁺ were not affected. Inhibition of CaN with CsA also led to a significant reduction in Ca²⁺ sensitivity of the channel at +50 mV; half‐maximal activation increased from 363 ± 16 nm Ca²⁺ in control to 515 ± 40 nm Ca²⁺ in the presence of CsA. Similar effects were observed on I_Cl(Ca) when a specific peptide fragment inhibitor of CaN (CaN‐AF, 5 μm) was dialysed into the cell via the pipette (500 nm Ca²⁺). Application of KN‐93 (10 μm), a specific inhibitor of CaMKII, enhanced I_Cl(Ca) in myocytes dialysed with 1 μm Ca²⁺ but produced no significant effect on this current when the cells were dialysed with 350 or 500 nm Ca²⁺. These results are consistent with the notion that in coronary arterial cells, the activity of Cl_Ca is enhanced by dephosphorylation of the channel or a closely associated regulatory protein. Moreover the balance of CaN and CaMKII regulating I_Cl(Ca) is dependent on the level of Ca²⁺ used to activate I_Cl(Ca).