Novel Kv3 glycoforms differentially expressed in adult mammalian brain contain sialylated N-glycans

Abstract

The N-glycan pool of mammalian brain contains remarkably high levels of sialylated N-glycans. This study provides the first evidence that voltage-gated K+ channels Kv3.1, Kv3.3, and Kv3.4, possess distinct sialylated N-glycan structures throughout the central nervous system of the adult rat. Electrophoretic migration patterns of Kv3.1, Kv3.3, and Kv3.4 glycoproteins from spinal cord, hypothalamus, thalamus, cerebral cortex, hippocampus, and cerebellum membranes digested with glycosidases were used to identify the various glycoforms. Differences in the migration of Kv3 proteins were attributed to the desialylated N-glycans. Expression levels of the Kv3 proteins were highest in cerebellum, whereas those of Kv3.1 and Kv3.3 were much lower in the other 5 regions. The lowest level of Kv3.1 was expressed in the hypothalamus, whereas the lowest levels of Kv3.3 were expressed in both thalamus and hypothalamus. The other regions expressed intermediate levels of Kv3.3, with spinal cord expressing the highest. The expression level of Kv3.4 in the hippocampus was slightly lower than that in cerebellum, and was closely followed by the other 4 regions, with spinal cord expressing the lowest level. We suggest that novel Kv3 glycoforms may endow differences in channel function and expression among regions throughout the central nervous system.

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

“…Kv3.1, 3.3 and 3.4 channels expressed in adult rat brain and spinal cord have sialylated N-glycans [20,22]. Additionally, we showed that the type of N-glycan attached to each of the Kv3 proteins is different in the various regions of the adult rat brain [22]. To further examine these sialylated N-glycans is quite challenging because expression levels of the Kv3 proteins are quite low.…”

“…Generally, these units consist of an initial α2,3-linked sialyl residue followed by α2,8-linked sialyl residues [2,4]. Kv3.1, 3.3 and 3.4 channels expressed in adult rat brain and spinal cord have sialylated N-glycans [20,22]. Additionally, we showed that the type of N-glycan attached to each of the Kv3 proteins is different in the various regions of the adult rat brain [22].…”

“…Kinetic selectivity was eliminated by extending the incubation periods of the reactions [33]. When sialylated N-glycans of the Kv3 [22] and Kv1 proteins were absent, immunoband shifts produced by the various exosialidases were absent. These results demonstrated that proteolysis did not contribute to immunoband shifts.…”

“…Additionally, the absolute . . conservation of the two N-glycosylation sites in the S1-S2 linker of the Kv3 proteins [20][21][22], along with the highly conserved site in the S1-S2 linker of the Kv1 subfamily members with the exception of Kv1.6 [17,52], argue for the importance of the sialyloligosaccharides in regulating neuronal excitability. Previously, we showed that the activation kinetics was slowed in unglycosylated Kv3.1 channels compared with those with simple type N-glycans [21].…”

N-Glycosylation in Regulation of the Nervous System

“…Glycosylation can affect cell excitability of neurons by modulating the function of various members of this channel superfamily, including channels that regulate membrane permeability for Na + , K + , and Ca 2+ ions (e.g., (Recio-Pinto et al 1990, Zhang et al 1999, Bennett 2002, Gong et al 2002, Watanabe et al 2003, Johnson et al 2004, Watanabe et al 2007, Schwetz et al 2010, Weiss et al 2013)). In mammals, N-glycosylation of voltage-gated Na + and K + channels was found to be regulated developmentally and in a cell-specific manner in the heart and the nervous system, suggesting that glycans participate in setting the distinct levels of excitability required in different cells and at various developmental stages (Castillo et al 1997, Tyrrell et al 2001, Schwalbe et al 2008, Montpetit et al 2009). …”