PKA-dependent regulation of mKv1.1 a mouse Shaker-like potassium channel gene when stably expressed in CHO cells

摘要

Potassium (K) channels are important regulators of cellular physiology and can themselves be modulated by phosphorylation. We have investigated the potential protein kinase A (PKA) regulation of mKv1.1, a mouse Shaker-like K channel gene, when it is expressed in stably transfected Chinese hamster ovary (CHO) cell lines. Whole-cell patch- clamp records show that expression of mKv1.1 gives rise to a rapidly activating, sustained K+ current, referred to classically as a delayed rectifier-type current. In order to study the effects of PKA, we compared cell lines transfected with mKv1.1 alone with lines cotransfected with both mKv1.1 and a plasmid encoding a dominant negative mutation in the regulatory subunit of PKA. These mutant regulatory subunits bind to endogenous catalytic subunits of PKA but do not respond to cAMP, thereby causing a chronic reduction in the basal PKA activity in these cells. We found that mKv1.1 current kinetics are unaltered but current density is 3.4-fold higher in the cell lines expressing mutant regulatory subunit than in lines expressing only mKv1.1. RNase protection assays indicate that levels of the specific RNA for mKv1.1 are increased almost twofold in the lines expressing mutant regulatory subunit over the lines expressing mKv1.1 only. Further, the levels of mKv1.1 protein, assayed using an mKv1.1 channel- specific antibody, are increased by almost a factor of 3 between the two types of cell lines. These results suggest that PKA can regulate mKv1.1 channel expression by changing steady-state levels of RNA and by other posttranscriptional mechanisms.