Transmembrane segments prevent surface expression of sodium channel Na v1.8 and promote calnexin-dependent channel degradation

Abstract

The voltage-gated sodium channel (Nav) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Nav1.8 is majorly localized intracellularly and its expression on the plasma membrane is regulated by exit from the endoplasmic reticulum (ER). Previous work has identified an ER-retention/retrieval motif in the first intracellular loop of Nav1.8, which prevents its surface expression. Here we report that the transmembrane segments of Nav1.8 also cause this channel retained in the ER. Using transferrin receptor and CD8α as model molecules, immunocytochemistry showed that the first, second, and third transmembrane segments in each domain of Nav1.8 reduced their surface expression. Alanine-scanning analysis revealed acidic amino acids as critical factors in the odd transmembrane segments. Furthermore, co-immunoprecipitation experiments showed that calnexin interacted with acidic amino acid-containing sequences through its transmembrane segment. Overexpression of calnexin resulted in increased degradation of those proteins through the ER-associated degradation pathway, whereas down-regulation of calnexin reversed the phenotype. Thus our results reveal a critical role and mechanism of transmembrane segments in surface expression and degradation of Nav1.8. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.