Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

Abstract

N‐Glycosylation is a cotranslational and post‐translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N‐glycosylation sequences of a voltage‐gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster‐migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N‐glycosylation of wild‐type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple‐type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild‐type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild‐type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1–S2 linker is topologically extracellular, and that N‐glycosylation influences the opening of the voltage‐dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

Identification of an Evolutionarily Conserved Extracellular Threonine Residue Critical for Surface Expression and Its Potential Coupling of Adjacent Voltage-sensing and Gating Domains in Voltage-gated Potassium Channels

“…4B, cells expressing WT HA 34 -tagged eYFPKv1.4 showed current densities (peak, 612 Ϯ 67 pA/pF (n ϭ 6); steady state, 517 Ϯ 50 pA/pF (n ϭ 6)) somewhat lower (p Ͻ 0.05, Tukey) than those obtained from cells transfected with WT eYFP-Kv1.4 ( Fig. 3) but typical for mutants bearing epitope tags at this position (30).…”

“…Nevertheless, such conservation may be fortuitous, and the requirement for Thr-330 in surface expression may be restricted to Kv1.4 or its close Kv1 series relatives. We therefore tested whether the conserved threonine might be a general requirement in Kv channels, using Kv3.1, which can form functional homotetramers but is more distantly related to, and unable to assemble with, Kv1.4 (5,33,34). Again, eYFP was fused to the amino terminus, and the resulting WT or mutant constructs (WT eYFP-Kv3.1 and eYFP-Kv3.1:T211A, respectively) were expressed in HEK293 cells.…”

The Axon–Dendrite Targeting of Kv3 (Shaw) Channels Is Determined by a Targeting Motif That Associates with the T1 Domain and Ankyrin G

“…HA-tagged Kv3.1a and Kv3.1b (Kv3.1aHA and Kv3.1bHA) were constructed by inserting an HA tag into the first extracellular loop after the second N-glycosylation site (Brooks et al, 2006). When expressed in HEK293 cells, Kv3.1aHA and Kv3.1bHA formed functional channels with activation potential at approximately Ϫ10 mV; the high activation threshold is a hallmark of Kv3 channels ( Fig.…”

Comparison of protein behavior between wild-type and G601S hERG in living cells by fluorescence correlation spectroscopy

“…G601S, in contrast, is subject to only core glycosylation [6], which may be responsible for the observed abnormal protein expression. Although the importance of glycosylation in protein expression and trafficking has been suggested [7,8], the effect of glycosylation on the expression and function of WT hERG and G601S is not fully understood. Moreover, in affected patients, G601S is co-expressed with the WT hERG form and behaves as a dominant negative.…”