Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary traje...