Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca(2+) Channels.
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.
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea.
Dong-Il
DI
Kim
Center for Proteome Biophysics, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea; Department of Physics, Pusan National University, Busan, South Korea.
Mooseok
M
Kang
Center for Proteome Biophysics, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea; Department of Physics, Pusan National University, Busan, South Korea.
Sangyeol
S
Kim
Center for Proteome Biophysics, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea; Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea.
Juhwan
J
Lee
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Yongsoo
Y
Park
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea; Center for Proteome Biophysics, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea. Electronic address: iksoochang@dgist.ac.kr.
Iksoo
I
Chang
Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu, South Korea. Electronic address: bcsuh@dgist.ac.kr.
Byung-Chang
BC
Suh
D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D055724:Electrophysiological Phenomena; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D042541:Intracellular Space / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D051381:Rats; D013816:Thermodynamics
C500963:Cacna1b protein, mouse; C491528:Cacnb2 protein, mouse; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate