Different Effects of Alkaline Phosphatase on HCN4 Channels in CHO Versus HEK Cells

Abstract

Hyperpolarization-activated, cyclic nucleotide-sensitive type 4 (HCN4) channels are regulated by cAMP, which can bind directly to a conserved cyclic nucleotide binding domain (CNBD) in the C-terminus. We previously found that the response of HCN4 channels to cAMP depends on the cellular context: cAMP binding to the CNBD shifts the midpoint activation voltage (V1/2) to more depolarized potentials when HCN4 is expressed in HEK cells, whereas cAMP has no effect on the V1/2 when HCN4 is expressed in CHO cells because the channels are already pre-activated, with a depolarized V1/2 in the absence of cAMP (Liao et al., 2012 J Gen Physiol 140(5):557). Here we have tested the hypothesis that differential phosphorylation may underlie the differences in HCN4 behavior in CHO versus HEK cells. We found that while alkaline phosphatase (AlkPhos) restored the hyperpolarized basal V1/2 of HCN4 in CHO cells, it did not restore the cAMP sensitivity of the channels. Quite different results were obtained in HEK cells, in which AlkPhos had little or no effect on the V1/2 of HCN4 in the absence of cAMP but, remarkably, was found to significantly reduce the shift in V1/2 in response to cAMP. These results indicate that dual regulation of HCN4 channels by cAMP and phosphorylation is complex and is sensitive to cellular context. And, phosphorylation alone is insufficient to account for the different behaviors of HCN4 channels in CHO versus HEK cells.