Direct cAMP Binding and PKA Phosphorylation Share a Common Gating Mechanism in HCN4 Channels

Abstract

Sympathetic regulation of HCN4 channels can occur via two cAMP-dependent pathways: either direct binding of cAMP to a cyclic nucleotide binding domain, or PKA phosphorylation of the distal C terminus. Here, we have investigated the energetic interactions between these two modulatory mechanisms. cDNA encoding wildtype or mutant mouse HCN4 channels was expressed in HEK293 cells, and the voltage-dependence of channel activation was determined in whole cell voltage clamp experiments. Intracellular dialysis of either PKA (20 U/ml) or cAMP (1 mM) shifted the activation midpoint (V1/2) of wild type HCN4 channels by ∼10 mV to more depolarized voltages. No additional shift was produced when both cAMP and PKA were introduced together, suggesting that the two modulators share a common final pathway in channel activation. To characterize further the independent effects of each modulator, we used PKA-insensitive (HCN4-Cx4) and cAMP-insensitive (HCN4-R669Q) mutant HCN4 channels. In PKA-insensitive HCN4-Cx4 channels, cAMP significantly shifted the voltage-dependence to more positive potentials, similar to its effect in wildtype HCN4 channels. This result demonstrates that cAMP modulation of HCN4 does not require PKA phosphorylation of the distal C-terminus. In contrast, PKA had no effect on the voltage-dependence of activation in the cAMP-insensitive HCN4-R669Q channels. Taken together, the data are consistent with a model in which direct binding of cAMP and PKA phosphorylation share a final common gating mechanism, however PKA modulation of HCN4 requires an unmodified cAMP binding domain.