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Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit.


ABSTRACT: The voltage-gated sodium channel ?2 subunit (Nav?2) is a physiological substrate of BACE1 (?-site APP cleaving enzyme) and ?-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Nav?2 by BACE1 and ?-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Nav?2.We found a major (147-148 L?M, where ? indicates the cleavage site) and a minor (144145 L?Q) BACE1 cleavage site in the extracellular domain of human Nav?2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Nav?2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Nav?2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Nav?2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Nav?2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of ?-secretase still actively cleaving the mutant proteins, Nav?2 cleavage products decreased by ~50% in cells expressing Nav?2 (147LM/VI) and ~75% in cells expressing Nav?2 (147LM/AA) as compared to cells expressing wildtype Nav?2.We identified a major (147-148 L?M) and a minor (144-145 L?Q) BACE1 cleavage site in human Nav?2. Our in vitro and cell-based results clearly show that the 147-148 L?M is the major BACE1 cleavage site in human Nav?2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

SUBMITTER: Gersbacher MT 

PROVIDER: S-EPMC3022600 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit.

Gersbacher Manuel T MT   Kim Doo Yeon DY   Bhattacharyya Raja R   Kovacs Dora M DM  

Molecular neurodegeneration 20101223


<h4>Background</h4>The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2.<h4>Results</h4>We found a major (147-148 L↓M, where ↓ indicates t  ...[more]

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