N-linked glycosylation sites of the motor protein prestin: effects on membrane targeting and electrophysiological function.

Abstract

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.