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

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