The immunoglobulin domain of the sodium channel ?3 subunit contains a surface-localized disulfide bond that is required for homophilic binding.

ABSTRACT: The ? subunits of voltage-gated sodium (Na(v)) channels possess an extracellular immunoglobulin (Ig) domain that is related to the L1 family of cell-adhesion molecules (CAMs). Here we show that in HEK293 cells, secretion of the free Ig domain of the ?3 subunit is reduced significantly when it is coexpressed with the full-length ?3 and ?1 subunits but not with the ?2 subunit. Using immunoprecipitation, we show that the ?3 subunit can mediate trans homophilic-binding via its Ig domain and that the ?3-Ig domain can associate heterophilically with the ?1 subunit. Evolutionary tracing analysis and structural modeling identified a cluster of surface-localized amino acids fully conserved between the Ig domains of all known ?3 and ?1 sequences. A notable feature of this conserved surface cluster is the presence of two adjacent cysteine residues that previously we have suggested may form a disulfide bond. We now confirm the presence of the disulfide bond in ?3 using mass spectrometry, and we show that its integrity is essential for the association of the full-length, membrane-anchored ?3 subunit with itself. However, selective reduction of this surface disulfide bond did not inhibit homophilic binding of the purified ?3-Ig domain in free solution. Hence, the disulfide bond itself is unlikely to be part of the homophilic binding site. Rather, we suggest that its integrity ensures the Ig domain of the membrane-tethered ?3 subunit adopts the correct orientation for productive association to occur in vivo.

SUBMITTER: Yereddi NR 

PROVIDER: S-EPMC3583845 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature