Glycosylation of GIRK1 at Asn119 and ROMK1 at Asn117 Has Different Consequences in Potassium Channel Function

Abstract

GIRK (G protein-gated inward rectifier K؉ channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn 119 , whereas GIRK4 was not glycosylated at Asn 132 . GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn 119 because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn 132 , either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn 119 does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn

Role of glycosylation in cell surface expression and stability of HERG potassium channels

“…N-linked glycosylation has also been reported to be required for the cell surface expression of ligand-gated channels including nicotinic acetylcholine receptor (16) and ATP purinergic receptors P2X1 and P2X2 (24 33). In contrast, other studies have shown that N-linked glycosylation is not required for the cell surface expression of cystic fibrosis transmembrane conductance regulator (17), cyclic nucleotide-gated channels (25), inward rectifier K ϩ channel GIRK1 (19), and voltagegated K ϩ channels such as Kv1.1 and Shaker (5,28). Previous biochemical studies of HERG channels in transfected cells and native cardiac tissues have shown that HERG channel protein is modified by N-linked glycosylation (7,20,21,37,38).…”

Gating of the shaker potassium channel is modulated differentially by N-glycosylation and sialic acids

“…For example, changes in glycosylation or sialylation altered the gating of potassium channels such as K v LQT1/mink, ROMK1, K v 1.1, and K v 4.3 [10,32,41,44]. Whereas other potassium channels such as squid K v 1 type and G-protein-coupled inward rectifying potassium channels appear to be unaffected by changes in glycosylation/sialylation [22,26]. These data suggest possible isoform specific differences in the sensitivity of K + channels to glycosylation.…”

Caveolin-3 and SAP97 form a scaffolding protein complex that regulates the voltage-gated potassium channel Kv1.5

“…However, the exact role may vary among different channels. For example, glycosylation reflects surface expression of HERG (23,30), alters the voltage dependence of activation of Kv1.1 and KvLQT1/minK, and modulates the open probability of ROMK1 (20,31). We and other investigators have shown that N-linked glycosylation is not essential for surface expression of Kv1.1 and Shaker channels in Xenopus oocytes (1).…”