Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Chen, Chunling; Calhoun, Judith G.; Zhang, Yanqing; Lopez-Santiago, Luis F.; Ning-na, Zhou; Davis, Tigwa H.; Salzer, James L.; Isom, Lori L.

doi:10.1074/jbc.m112.397646

Cited by 66 publications

(80 citation statements)

References 30 publications

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“…Coexpression of Na V β2-and Na V β4-subunits, as well as MTSET treatment, produces the same effect; namely, the channels are protected against block by GVIIJ SSG . Unlike Na V β1 and Na V β3, which are noncovalently attached to their Na V 1-subunit, each of Na V β2 and Na V β4 is linked to its Na V 1-subunit via a disulfide bond (1,6). Thus, it is tempting to speculate that C910 of rNa V 1.2 might be disulfide-linked to its counterpart in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…*, The first bar is lower than the second bar, P < 0.0002; heights of the second, fourth, and fifth bars are not significantly different from each other (P > 0.5). (C) Hypothesized states of site 8, wherein P represents the peptide either as PSH (i.e., GVIIJ SH ) or as PS-SR where Cys24 is disulfide bonded to R, which is either cysteine (GVIIJ SSC Na V β2 or Na V β4, a possibility also raised by Chen et al for Na V β2 based on other considerations (6).…”

Section: Discussionmentioning

confidence: 99%

“…Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1,6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).…”

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confidence: 99%

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A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

Gajewiak¹,

Azam²,

Imperial³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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A cone snail venom peptide, μO §-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligandbinding site. μO §-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG ). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V 1.2. A mutant channel of rNa V 1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 3 -fold less sensitive to GVIIJ SSG than was wild-type rNa V 1.2. In contrast, although rNa V 1.5 was >10 4 -fold less sensitive to GVIIJ SSG than Na V 1.2, an rNa V 1.5 mutant with a cysteine in the homologous location, rNa V 1.5[L869C], was >10 3 -fold more sensitive than wildtype rNa V 1.5. The susceptibility of rNa V 1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa V β2 or rNa V β4, but not that of rNa V β1 or rNa V β3, protected rNa V 1.1 to -1.7 (excluding Na V 1.5) against block by GVIIJ SSG . Thus, GVIIJrelated peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na V β-and Na V α-subunits are present in native neurons.oltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable tissues. Each VGSC is composed of a pore-and voltage sensor-bearing α-subunit and one or more auxiliary β-subunits. Mammals have nine α-subunit isoforms (Na V 1.1 to -1.9) and four β-subunit isoforms (Na V β1 to -β4) (1). An Na V 1 has about 2,000-aa residues arranged in four homologous domains, where each domain has six transmembrane spanning segments with an extracellular "pore" loop between segments 5 and 6 (1, 2); furthermore, each Na V 1 has about a dozen extracellular cysteine residues, all located in or near the pore loops. For the most part, not much is known about these cysteines (including whether they are disulfide bonded).Na V β-subunits can affect the function and cellular localization of Na V 1s (1, 3-5). Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1, 6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).Toxins that target VGSCs have been invaluable for probing the structure and function of these channels. Venoms are a rich source of such toxins. For example, in Conus snails, four families of neuroactive peptides have been characterized that target VGSCs:...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

Gajewiak¹,

Azam²,

Imperial³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…3B). Strikingly, the position of 58 Cys in β4 corresponds to that of the Cys ( 26 Cys) thought to be involved in linking β2 to Na v 1.1 (40). Here, removing the covalent bond with Na v 1.1 by mutating 26 Cys to Ala results in an altered subcellular localization pattern of β2.…”

Section: Significancementioning

confidence: 99%

Crystallographic insights into sodium-channel modulation by the β4 subunit

Gilchrist

Das

Petegem

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

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Voltage-gated sodium (Na v ) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, β-subunits modify Na v channel function and cause debilitating disorders when mutated. While investigating whether β-subunits also influence ligand interactions, we found that β4 dramatically alters toxin binding to Na v 1.2. To explore these observations further, we solved the crystal structure of the extracellular β4 domain and identified 58 Cys as an exposed residue that, when mutated, eliminates the influence of β4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of β4. Disrupting this bridge by introducing a β1 mutation implicated in epilepsy repositions the 58 Cys-containing loop and disrupts β4 modulation of Na v 1.2. Overall, the principles emerging from this work (i) help explain tissuedependent variations in Na v channel pharmacology; (ii) enable the mechanistic interpretation of β-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant β-subunit behavior.voltage-gated sodium channel | beta4 subunit | ProTx-II | X-ray structure | disease mutations

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“…Na þ channels also interact with the accessory b-subunits Navb1, Navb2, and Navb4 (Chen et al 2002(Chen et al , 2004Buffington and Rasband 2013). The b-subunits are covalently linked to Na þ channels through an extracellular disulfide bond (Chen et al 2012;Buffington and Rasband 2013) to promote the surface expression of Na þ channels and change their biophysical properties. In addition to Na þ channels, nodes of Ranvier are also enriched in K þ channels (Cooper 2011).…”

Section: Nodes Of Ranviermentioning

confidence: 99%

The Nodes of Ranvier: Molecular Assembly and Maintenance

Rasband

Peles

2015

Cold Spring Harb Perspect Biol

161

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Action potential (AP) propagation in myelinated nerves requires clustered voltage gated sodium and potassium channels. These channels must be specifically localized to nodes of Ranvier where the AP is regenerated. Several mechanisms have evolved to facilitate and ensure the correct assembly and stabilization of these essential axonal domains. This review highlights the current understanding of the axon intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the peripheral nervous system (PNS) and central nervous system (CNS).A xons conduct electrical signals, called action potentials (APs), among neurons in a circuit in response to sensory input, and between motor neurons and muscles. In mammals and other vertebrates, many axons are myelinated. Myelin, made by Schwann cells and oligodendrocytes in the peripheral nervous system (PNS) and central nervous system (CNS), respectively, is a multilamellar sheet of glial membrane that wraps around axons to increase transmembrane resistance and decrease membrane capacitance. Although myelin is traditionally viewed as a passive contributor to nervous system function, it is now recognized that myelinating glia also play many active roles including regulation of axon diameter, axonal energy metabolism, and the clustering of ion channels at gaps in the myelin sheath called nodes of Ranvier. Together, the active and passive properties conferred on axons by myelin, result in axons with high AP conduction velocities, low metabolic demands, and reduced space requirements as compared with unmyelinated axons. Thus, myelin and the clustering of ion channels in axons permitted the evolution of the complex nervous systems found in vertebrates. This review highlights the current understanding of the axonal intrinsic and glial extrinsic mechanisms that control the formation and maintenance of the nodes of Ranvier in both the PNS and CNS.

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Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Cited by 66 publications

References 30 publications

A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

Crystallographic insights into sodium-channel modulation by the β4 subunit

The Nodes of Ranvier: Molecular Assembly and Maintenance

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