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Molecular Basis of the Membrane Interaction of the beta 2e Subunit of Voltage-Gated Ca2+ Channels

Title
Molecular Basis of the Membrane Interaction of the beta 2e Subunit of Voltage-Gated Ca2+ Channels
Authors
Kim, DI[Kim, Dong-Il]Kang, M[Kang, Mooseok]Kim, S[Kim, Sangyeol]Lee, J[Lee, Juhwan]Park, Y[Park, Yongsoo]Chang, I[Chang, Iksoo]Suh, BC[Suh, Byung-Chang]
DGIST Authors
Kim, DI[Kim, Dong-Il]; Kang, M[Kang, Mooseok]; Kim, S[Kim, Sangyeol]; Lee, J[Lee, Juhwan]; Chang, I[Chang, Iksoo]Suh, BC[Suh, Byung-Chang]
Issue Date
2015-09-01
Citation
Biophysical Journal, 109(5), 922-935
Type
Article
Article Type
Article
ISSN
0006-3495
Abstract
The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels. © 2015 Biophysical Society.
URI
http://hdl.handle.net/20.500.11750/1711
DOI
10.1016/j.bpj.2015.07.040
Publisher
Cell Press
Related Researcher
  • Author Chang, Ik Soo Theoretical and Computational Biophysics Laboratory
  • Research Interests
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Collection:
New BiologyETC1. Journal Articles
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Emerging Materials ScienceETC1. Journal Articles
Brain and Cognitive SciencesTheoretical and Computational Biophysics Laboratory1. Journal Articles


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