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1.
Open Biol ; 5(1): 140192, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25567098

ABSTRACT

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated ß subunits. The ß subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human ß3 and ß4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that ß3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some ß subunits.


Subject(s)
Voltage-Gated Sodium Channel beta Subunits/chemistry , Action Potentials , Amino Acid Sequence , Animals , Evolution, Molecular , Humans , Ion Channel Gating , Molecular Sequence Data , Voltage-Gated Sodium Channel beta Subunits/genetics , Voltage-Gated Sodium Channel beta Subunits/metabolism
2.
FASEB J ; 27(2): 568-80, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23118027

ABSTRACT

The ß 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 ß3 subunit is reduced significantly when it is coexpressed with the full-length ß3 and ß1 subunits but not with the ß2 subunit. Using immunoprecipitation, we show that the ß3 subunit can mediate trans homophilic-binding via its Ig domain and that the ß3-Ig domain can associate heterophilically with the ß1 subunit. Evolutionary tracing analysis and structural modeling identified a cluster of surface-localized amino acids fully conserved between the Ig domains of all known ß3 and ß1 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 ß3 using mass spectrometry, and we show that its integrity is essential for the association of the full-length, membrane-anchored ß3 subunit with itself. However, selective reduction of this surface disulfide bond did not inhibit homophilic binding of the purified ß3-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 ß3 subunit adopts the correct orientation for productive association to occur in vivo.


Subject(s)
Voltage-Gated Sodium Channel beta-3 Subunit/chemistry , Amino Acid Sequence , Binding Sites , Disulfides/chemistry , Evolution, Molecular , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Voltage-Gated Sodium Channel beta-1 Subunit/chemistry , Voltage-Gated Sodium Channel beta-1 Subunit/genetics , Voltage-Gated Sodium Channel beta-1 Subunit/metabolism , Voltage-Gated Sodium Channel beta-3 Subunit/genetics , Voltage-Gated Sodium Channel beta-3 Subunit/metabolism
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