Voltage-gated sodium channels assemble and gate as dimers

Clatot, Jérôme; Hoshi, Malcolm; Wan, Xiaoping; Liu, Haiyan; Jain, Ankur; Shinlapawittayatorn, Krekwit; Marionneau, Céline; Ficker, Eckhard; Ha, Taekjip; Deschênes, Isabelle

doi:10.1038/s41467-017-02262-0

Cited by 111 publications

(209 citation statements)

References 38 publications

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“…Previous evidence suggests that heterologously expressed Nav1.5 α‐subunits can assemble as dimers . The epitope recognized by the antibody used in our STORM experiments is located at the cytoplasmic C‐terminus of the Nav1.5 α‐subunit.…”

Section: Discussionmentioning

confidence: 76%

“…26 Previous evidence suggests that heterologously expressed Nav1.5 α-subunits can assemble as dimers. [27][28][29][30][31] The epitope recognized by the antibody used in our STORM experiments is located at the cytoplasmic C-terminus of the Nav1.5 α-subunit. By comparison with other Nav channels whose near atomic-resolution structure has been resolved, it is likely that this epitope lie close to or underneath the central pore of the channel, albeit with some flexibility.…”

Section: Discussionmentioning

confidence: 99%

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Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

et al. 2020

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Voltage‐gated sodium channels comprise an ion‐selective α‐subunit and one or more associated β‐subunits. The β3‐subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart‐specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full‐length β3‐subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3‐subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α‐subunit and the effects upon these of the β3‐subunits. The oligomeric status of the Nav1.5 α‐subunit in vivo, with or without the β3‐subunit, has not been previously investigated. Using super‐resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α‐subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3‐subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest‐neighbor distances between Nav1.5 α‐subunits. However, when assayed by PLA, the β3‐subunit increases the number of PLA‐positive signals generated by anti‐(Nav1.5 α‐subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3‐subunit introduces a significant change in the relative alignment of individual Nav1.5 α‐subunits, but the clustering itself depends on other factors. We also show that these structural and higher‐order changes induced by the β3‐subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α‐subunits. Our data provide new insights into the role of the β3‐subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

et al. 2020

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“…One cell, however, displayed a strong preference for doublet channel openings after application of ISO (Fig. G and H ), suggestive of a co‐operative dimer of Ca V 1.2 channels, reminiscent of the recently reported dimeric preference of co‐operatively gating Na V 1.5 channels (Clatot et al ., ). These data strongly support the hypothesis that ISO promotes co‐operative interactions between Ca V 1.2 channels in ventricular myocytes.…”

Section: Resultsmentioning

confidence: 97%

β‐adrenergic‐mediated dynamic augmentation of sarcolemmal Ca_V1.2 clustering and co‐operativity in ventricular myocytes

Ito

Hannigan

Ghosh

et al. 2019

The Journal of Physiology

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Key points Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type Ca V 1.2 channel activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. Ca V 1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of Ca V 1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca 2+ influx. On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized Ca V 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling. Abstract Voltage‐dependent L‐type Ca V 1.2 channels play an indispensable role in cardiac excitation–contraction coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced Ca V 1.2 activity, resulting in increased Ca 2+ influx into ventricular myocytes and a positive inotropic response. Ca V 1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca 2+ ‐dependent co‐operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca 2+ influx and augments myocyte Ca 2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter Ca V 1.2 channel clustering to facilitate co‐operative channel interactions and elevate Ca 2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced Ca V 1.2–Ca V 1.2 physical interactions. Super‐resolution nanoscopy and dynamic channel tracking indicate tha...

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“…10,14 On the other hand, absence epilepsy was observed in a family with the heterozygous protein truncating variant p.Asn544fs*39, 6 and focal epilepsy with mild ID was observed for the protein truncation variant p.Arg1820*0. 30 In this case, LOF alleles that produce full length protein could have a dominant negative effect in heterozygotes, resulting in <50% residual activity and a more severe phenotype than that of LOF alleles encoding truncated or unstable proteins. 18,29 It is also possible that missense variants that appear to cause complete LOF in vitro may actually retain some activity in vivo that contributes to the relatively mild phenotype in the monoallelic (heterozygous) parents.…”

Section: Discussionmentioning

confidence: 99%

Biallelic inherited SCN8A variants, a rare cause of SCN8A‐related developmental and epileptic encephalopathy

Wengert

Tronhjem²,

Wagnon

et al. 2019

Epilepsia

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Objective: Monoallelic de novo gain-of-function variants in the voltage-gated sodium channel SCN8A are one of the recurrent causes of severe developmental and epileptic encephalopathy (DEE). In addition, a small number of de novo or inherited monoallelic loss-of-function variants have been found in patients with intellectual disability, autism spectrum disorder, or movement disorders. Inherited monoallelic variants causing either gain or loss-of-function are also associated with less severe conditions such as benign familial infantile seizures and isolated movement disorders. In all three categories, the affected individuals are heterozygous for a SCN8A variant in combination with a wild-type allele. In the present study, we describe two unusual families with severely affected individuals who inherited biallelic variants of SCN8A. Methods: We identified two families with biallelic SCN8A variants by diagnostic gene panel sequencing. Functional analysis of the variants was performed using voltage clamp recordings from transfected ND7/23 cells. Results: We identified three probands from two unrelated families with DEE due to biallelic SCN8A variants. Each parent of an affected individual carried a single heterozygous SCN8A variant and exhibited mild cognitive impairment without seizures. In both families, functional analysis demonstrated segregation of one allele with complete loss-of-function, and one allele with altered biophysical properties consistent with partial loss-of-function. Significance: These studies demonstrate that SCN8A DEE may, in rare cases, result from inheritance of two variants, both of which exhibit reduced channel activity. In these families, heterozygosity for the dominant variants results in less severe disease than biallelic inheritance of two variant alleles. The clinical consequences of variants 2278 | WENGERT ET al.

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Voltage-gated sodium channels assemble and gate as dimers

Cited by 111 publications

References 38 publications

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

β‐adrenergic‐mediated dynamic augmentation of sarcolemmal Ca_V1.2 clustering and co‐operativity in ventricular myocytes

Biallelic inherited SCN8A variants, a rare cause of SCN8A‐related developmental and epileptic encephalopathy

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Voltage-gated sodium channels assemble and gate as dimers

Cited by 111 publications

References 38 publications

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary β3‐subunit

β‐adrenergic‐mediated dynamic augmentation of sarcolemmal CaV1.2 clustering and co‐operativity in ventricular myocytes

Biallelic inherited SCN8A variants, a rare cause of SCN8A‐related developmental and epileptic encephalopathy

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β‐adrenergic‐mediated dynamic augmentation of sarcolemmal Ca_V1.2 clustering and co‐operativity in ventricular myocytes