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Determining the molecular basis of voltage sensitivity in membrane proteins.

ABSTRACT: Voltage-sensitive membrane proteins are united by their ability to transform changes in membrane potential into mechanical work. They are responsible for a spectrum of physiological processes in living organisms, including electrical signaling and cell-cycle progression. Although the mechanism of voltage-sensing has been well characterized for some membrane proteins, including voltage-gated ion channels, even the location of the voltage-sensing elements remains unknown for others. Moreover, the detection of these elements by using experimental techniques is challenging because of the diversity of membrane proteins. Here, we provide a computational approach to predict voltage-sensing elements in any membrane protein, independent of its structure or function. It relies on an estimation of the propensity of a protein to respond to changes in membrane potential. We first show that this property correlates well with voltage sensitivity by applying our approach to a set of voltage-sensitive and voltage-insensitive membrane proteins. We further show that it correctly identifies authentic voltage-sensitive residues in the voltage-sensor domain of voltage-gated ion channels. Finally, we investigate six membrane proteins for which the voltage-sensing elements have not yet been characterized and identify residues and ions that might be involved in the response to voltage. The suggested approach is fast and simple and enables a characterization of voltage sensitivity that goes beyond mere identification of charges. We anticipate that its application before mutagenesis experiments will significantly reduce the number of potential voltage-sensitive elements to be tested.

SUBMITTER: Kasimova MA

PROVIDER: S-EPMC6168238 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Determining the molecular basis of voltage sensitivity in membrane proteins.

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