N-Linked Glycosylation Regulates CALHM1 Channel Function and Subcellular Localization

Abstract

Calcium homeostasis modulator 1 (CALHM1) was discovered as a susceptibility gene for late-onset Alzheimer's disease. CALHM1 has been established as a pore-forming subunit of a homohexameric voltage-gated, non-selective ion channel and its important physiological roles including neuronal excitability and taste perception have also been revealed. However, other modes of CALHM1 channel regulation including ones mediated by post-translational modifications remain to be elucidated. On a western blot, heterologously expressed mouse CALHM1 (mCALHM1) immunosignal appears as three bands with one band at the size of a mCALHM1 monomer, approximately 37 kDa, and the other two bands at higher molecular weights, suggesting post-translational modifications on CALHM1. The two higher molecular weight bands disppeared after peptide-N-glycosidase F treatment, demonstrating that mCALHM1 is glycosylated at asparagine (N) residue(s). Point mutation studies determined that a conserved N139 is the only N-glycosylation site on mCALHM1. The two glycosylated forms of mCALHM1 showed different sinsitivities to endoglycosidase H, demonstrating that conversion of the acquired N-linked glycan chain from high-mannose type to complex type takes place. Furthermore, by using chemical and enzymatic reagents, point mutation studies, and glycosylation mutant cell lines, we examined roles of acquisition and conversion of the N-linked glycan on function and subcellular localization of mCALHM1 channel. Our data provide insights into a novel regulation of CALHM1 channel by N-linked glycosylation.