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Development and Application of an MRM Method for Simultaneous Quantification of Sodium Channels Na v 1.1, Na v 1.2 and Na v 1.6 in Stable HEK293 cell lines, and Solubilized Membrane Proteins from Rodents and Human Brain Tissues.
  • +8
  • Luis Sojo,
  • Raibow Kwan,
  • Prerna Das,
  • Neelan Gerrebos,
  • Jenny Li,
  • Xin Yin Wang,
  • Gina DeBoer,
  • Vanessa Emnacen-Pankhurst,
  • Sophia Lin,
  • Raymond Feng,
  • Sam Goodchild
Luis Sojo
Xenon Pharmaceuticals Inc

Corresponding Author:[email protected]

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Raibow Kwan
Xenon Pharmaceuticals Inc
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Prerna Das
Xenon Pharmaceuticals Inc
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Neelan Gerrebos
Xenon Pharmaceuticals Inc
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Jenny Li
Xenon Pharmaceuticals Inc
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Xin Yin Wang
Xenon Pharmaceuticals Inc
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Gina DeBoer
Xenon Pharmaceuticals Inc
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Vanessa Emnacen-Pankhurst
Xenon Pharmaceuticals Inc
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Sophia Lin
Xenon Pharmaceuticals Inc
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Raymond Feng
Xenon Pharmaceuticals Inc
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Sam Goodchild
Xenon Pharmaceuticals Inc
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Abstract

Rationale Na v1.1, 1.2 and 1.6 are transmembrane proteins acting as voltage gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods Unique peptides for Na v1.1, Na v1.2 and Na v1.6 were selected as quantotropic peptides for each protein and used for their quantification in plasma membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry. Results Na v 1.1, 1.2 and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1±0.2, 6.4±1.2 and 4.0±0.6 fmole/µg membrane protein respectively. Na v1.2 showed the highest expression, with approximately 3 times higher (p<0.003) in rodents than in human at 3.05±0.57, 3.35±0.56 in mouse and rat brains and 1.09±0.27 fmole/µg in human, respectively. Both Na v1.1 and 1.6 expressions were much lower than Na v1.2, with approximately 40% less expression in human Na v1.1 when compared with rodents Na v1.1 at 0.49±0.1 (mouse), 0.43±0.3 (rat), and 0.28±0.04 (human); while Na v1.6 was approximately 60% less expression in human when compared to rodents at 0.27±0.09 (mouse), 0.26±0.06 (rat) and 0.11±0.02 (human) fmole/µg membrane proteins. Conclusions MRM was used to quantify sodium channels Na v1.1, 1.2 and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mouse, rat, and human. We found significant differences in the expression of these channels in mouse, rat, and human brains. Na v expression ranking among the three species was Na v1.2 >> Na v1.1> Na v1.6, with the human brain expressing much lower concentrations overall in comparison to rodents.
30 Jun 2023Submitted to Rapid Communications in Mass Spectrometry
30 Jun 2023Submission Checks Completed
30 Jun 2023Assigned to Editor
30 Jun 2023Review(s) Completed, Editorial Evaluation Pending
02 Jul 2023Reviewer(s) Assigned
21 Jul 2023Editorial Decision: Revise Major
12 Sep 20231st Revision Received
12 Sep 2023Submission Checks Completed
12 Sep 2023Assigned to Editor
12 Sep 2023Review(s) Completed, Editorial Evaluation Pending
15 Sep 2023Reviewer(s) Assigned
01 Oct 2023Editorial Decision: Revise Minor
13 Oct 20232nd Revision Received
16 Oct 2023Assigned to Editor
16 Oct 2023Submission Checks Completed
16 Oct 2023Review(s) Completed, Editorial Evaluation Pending
22 Oct 2023Editorial Decision: Accept