Abstract
Hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) belongs to the Cyclic Nucleotide-Gated (CNG) channel superfamily, which has been found to control pacemaker activity in the heart and brain. HCN2 was shown to undergo N-linked glycosylation. However the effect of the glycosylation on the expression and function of HCN2 channels is still under debate. In this study, we use biochemical modification, confocal microscopy and flux assay to investigate the role of N-glycosylation on the membrane trafficking and function of HCN2 channels. We showed that HCN2 mutant N380Q is not N-glycosylated. Confocal microscopy and Western Blot are used to ascertain the subcellular localization of N380Q mutant. By using PNGase-F digestion, WGA lectin beads isolation, and antibody affinity purification from plasma membranes, we showed that N-glycosylation on HCN2 channels is heterogeneous. One step further, we reconstituted HCN2 channels onto liposomes and established a cation flux assay to study the function of reconstituted HCN2 wild type and N380Q mutant. The result generated in this study will answer the question of whether the N-glycosylation is critical for the function of HCN2 channels. The method developed here will provide a platform, which could be applied for studying the functions of other CNG channels.
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