© 1995 American Heart Association, Inc.
Articles |
Unitary Chloride Channels Activated by Protein Kinase C in Guinea Pig Ventricular Myocytes
From the Department of Physiology, University of Nevada School of Medicine, Reno.
Correspondence to Dr. Mei Lin Collier, Department of Physiology, University of Nevada School of Medicine, Anderson Medical Science Building, Reno, NV 89557-0046.
Abstract Recent evidence suggests that protein kinase A (PKA)-activated Cl- channels in heart are encoded by an isoform of the epithelial cystic fibrosis transmembrane conductance regulator gene (CFTR). Macroscopic current measurements indicate that a similar time-independent Cl- conductance can be activated through a protein kinase C (PKC)-dependent pathway in guinea pig and feline ventricle. However, it is presently not clear whether PKC is activating the same population of channels as PKA or a separate class of Cl- channels, even though the regulatory (R) domain of CFTR is known to contain consensus phosphorylation sites for both PKA and PKC. In the present study we directly compare the properties of single Cl- channels activated by PKC and PKA in cell-attached patches of guinea pig ventricular myocytes. Pipette and bath solutions contained N-methyl-D-glucamine and Cs+ or tetraethylammonium as substitutes for Na+ and K+, respectively, and Cl- concentration in the patch pipette was either 150 mmol/L or 40 mmol/L. Bath application of phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate (PDBu or PMA; 50 nmol/L), activators of PKC, resulted in the appearance of unitary Cl- channels with a mean conductance of 9.31±0.94 pS (n=8) and 8.8 pS (n=2), respectively, and reversal potentials were close to predicted ECl. Addition of staurosporine (500 nmol/L) reduced open probability (Po) of channels activated by PDBu. Bath application of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 µmol/L) resulted in the activation of Cl- channels with a conductance (mean 8.76±0.67 pS, n=3) similar to those activated by PDBu. Open probability (Po) of both PKC- and PKA-activated Cl- channels exhibited voltage independence and both were insensitive to block by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In patches containing channels preactivated by PDBu, subsequent bath application of IBMX (500 µmol/L) resulted in an increase in Po without any evidence for the activation of a separate population of channels with a different unitary conductance. These results suggest either that PKC can activate cardiac CFTR Cl- channels and that once activated by PKC, the channels can be further modulated by PKA or that PKC and PKA activate separate populations of Cl- channels with similar conductance and kinetic properties.
Key Words: Cl- channels • protein kinase C • guinea pig ventricle • CFTR • cardiac electrophysiology
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