Functional Expression of Rat Nav1.6 Voltage-Gated Sodium Channels in HEK293 Cells: Modulation by the Auxiliary β1 Subunit

Background: Genetic variants in voltage-gated sodium channels (Nav) encoded by SCNXA genes, responsible for INa, and Kv4.3 channels encoded by KCND3, responsible for the transient outward current (Ito), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that Kv4.3 and Nav variants regulate each other’s function, thus modulating INa/Ito balance in cardiomyocytes and INa/I(A) balance in neurons. Methods: Bicistronic and other constructs were used to express WT or variant Nav1.5 and Kv4.3 channels in HEK293 cells. INa and Ito were recorded. Results: SCN5A variants associated with BrS reduced INa, but increased Ito. Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of Ito, significantly reduced INa, whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of Ito significantly increased INa. Auxiliary subunits Navβ1, MiRP3 and KChIP2 also modulated INa/Ito balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase Kv4.3 cell-surface expression. Conclusion: Nav and Kv4.3 channels modulate each other’s function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.

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Functional expression of voltage-gated sodium channels Nav1.5 in human breast caner cell line MDA-MB-231

Journal of Huazhong University of Science and Technology [Medical Sciences] ◽

10.1007/s11596-009-0113-5 ◽

2009 ◽

Vol 29 (1) ◽

pp. 64-67 ◽

Cited By ~ 8

Author(s):

Rui Gao ◽

Jing Wang ◽

Yi Shen ◽

Ming Lei ◽

Zehua Wang

Keyword(s):

Cell Line ◽

Sodium Channels ◽

Functional Expression ◽

Human Breast ◽

Voltage Gated ◽

Voltage Gated Sodium Channels ◽

Breast Caner

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Non-SUMOylated CRMP2 decreases NaV1.7 currents via the endocytic proteins Numb, Nedd4-2 and Eps15

Molecular Brain ◽

10.1186/s13041-020-00714-1 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Kimberly Gomez ◽

Dongzhi Ran ◽

Cynthia L. Madura ◽

Aubin Moutal ◽

Rajesh Khanna

Keyword(s):

Sodium Channels ◽

Mechanical Allodynia ◽

Neuronal Excitability ◽

Growth Factor Receptor ◽

Functional Expression ◽

Voltage Gated ◽

Female Mice ◽

Voltage Gated Sodium Channels ◽

Endocytic Proteins

AbstractVoltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

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Studies on CRMP2 SUMOylation-deficient transgenic mice reveal novel insights into the trafficking of NaV1.7 channels

10.1101/2020.09.29.318774 ◽

2020 ◽

Author(s):

Kimberly Gomez ◽

Dongzhi Ran ◽

Cynthia L. Madura ◽

Aubin Moutal ◽

Rajesh Khanna

Keyword(s):

Sodium Channels ◽

Neuronal Excitability ◽

Growth Factor Receptor ◽

Functional Expression ◽

Male And Female ◽

Voltage Gated ◽

Female Mice ◽

Voltage Gated Sodium Channels ◽

Mediator Protein

AbstractVoltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel NaV1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). If not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2K374A/K374A) female mice have reduced NaV1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male and female CRMP2K374A/K374A mice, increased pain sensitivity in allodynia-resistant animals. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

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