Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Winterstein, Laura-Marie; Kukovetz, Kerri; Rauh, Oliver; Turman, Daniel L.; Braun, Christian; Moroni, Anna; Schroeder, Indra; Thiel, Gerhard

doi:10.1085/jgp.201711904

Cited by 35 publications

(46 citation statements)

References 48 publications

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“…The Kmpv PL1 , Kmpv SP1 and Kmpv 1 proteins were synthetized cell-free according to the manufacturer's instructions (MembraneMax TM HN Protein Expression Kit (Invitrogen, Carlsbad (CA) USA) as reported previously [21]. In vivo synthesis occurred on a shaker with 1000 rpm at 37°C for 1.5 h in the presence of nanodiscs (ND) with 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) lipids.…”

Section: Methodsmentioning

confidence: 99%

A small viral potassium ion channel with an inherent inward rectification

et al. 2019

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Some algal viruses have coding sequences for proteins with structural and functional characteristics of pore modules of complex K + channels. Here we exploit the structural diversity among these channel orthologs to discover new basic principles of structure/function correlates in K + channels. The analysis of three similar K + channels with ≤ 86 amino acids (AA) shows that one channel (Kmpv 1) generates an ohmic conductance in HEK293 cells while the other two (Kmpv SP1 , Kmpv PL1) exhibit typical features of canonical Kir channels. Like Kir channels, the rectification of the viral channels is a function of the K + driving force. Reconstitution of Kmpv SP1 and Kmpv PL1 in planar lipid bilayers showed rapid channel fluctuations only at voltages negative of the K + reversal voltage. This rectification was maintained in KCl buffer with 1 mM EDTA, which excludes blocking cations as the source of rectification. This means that rectification of the viral channels must be an inherent property of the channel. The structural basis for rectification was investigated by a chimera between rectifying and non-rectifying channels as well as point mutations making the rectifier similar to the ohmic conducting channel. The results of these experiments exclude the pore with pore helix and selectivity filter as playing a role in rectification. The insensitivity of the rectifier to point mutations suggests that tertiary or quaternary structural interactions between the transmembrane domains are responsible for this type of gating.

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Section: Methodsmentioning

confidence: 99%

A small viral potassium ion channel with an inherent inward rectification

et al. 2019

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“…Detergent-solubilized MPs can be either used directly for functional analysis or may be reconstituted into liposomes by mixing with artificial lipids followed by detergent removal [9,82,83]. Alternatively, nanodiscs (NDs) and liposome-based reconstitution are detergent-free strategies where NDs and liposomes, prepared and characterized externally, could be supplemented directly into the reaction mixture for the reconstitution [9,84]. A detailed review of the CF synthesis of MPs and the usage of solubilization supplements for isolation and functional analysis is presented in the literature [9].…”

Section: Cell-free Systems For the Synthesis Of Membrane Proteinsmentioning

confidence: 99%

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

et al. 2020

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Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. Key PointsCell-free protein synthesis has the potential to become an alternative method for the rapid and highly parallel expression of a diverse range of proteins.Cell-free systems utilize the translation machinery of the cells, bypassing the constraints of the cell membrane and thus offer openness and configurational flexibility even for the synthesis of difficult-to-express proteins.In comparison to traditional approaches, cell-free systems can be time-saving, from initial synthesis to downstream applications.

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“…Winterstein et al . incorporated functional K + channels and F − channels into nanoscale lipid bilayers or nanodiscs . The study provided a fast and reliable method for functional analysis of these ion channels.…”

Section: Incorporation Of Diffusion Channels Into Synthetic Systemsmentioning

confidence: 99%

“…. There are some limitations involved in incorporation strategies such as functionality of these proteins, similarity of synthetic system with the natural environment of lipid bilayer in the cell membrane .…”

Section: Incorporation Of Diffusion Channels Into Synthetic Systemsmentioning

confidence: 99%

“…Amphiphilic block copolymers have shown their ability to be an excellent alternative for lipids in terms of stability, flexibility, and robustness [55]. Winterstein et al incorporated functional K + channels and F − channels into nanoscale lipid bilayers or nanodiscs [59]. The study provided a fast and reliable method for functional analysis of these ion channels.…”

Section: Incorporation Of Diffusion Channels Into Synthetic Systemsmentioning

confidence: 99%

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Biomimetic and bioinspired membranes for water purification: A critical review and future directions

Wagh

Escobar

2019

Env Prog and Sustain Energy

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Various types of channel proteins, broadly named porins, present in the cell membrane of gram‐negative bacteria have specific functionalities depending on their selectivity toward different nutrients or toward water. The high selectivity of porins has led to their incorporation into synthetic systems, in a field called biomimetics. An example is the addition of water channel proteins, or aquaporins, to polymeric separations membranes in order to enhance their performance in terms of selectivity and permeability. The concept of incorporating aquaporins into synthetic membranes has been studied for the last 10 years; however, there are still limitations such as costs, alignment into the membrane assembly, and scalability of membrane fabrication. Therefore, in recent years, there has been an increase in the study of synthesizing molecules with similar structure–function relationships of porins. These artificial channels attempt to mimic the biological porins, while being synthesized using simpler chemistry, being solvent compatible, and requiring less space on the membrane surface which helps to incorporate more channels into the membrane assembly. In summary, the future of biomimetic and bioinspired membranes depends on the design strategies, level of nature imitation, and the performance of these systems on a commercial scale. © 2019 American Institute of Chemical Engineers Environ Prog, 38:e13215, 2019

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Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Abstract: Ion channel proteins can be in vitro translated into nanoscale lipid bilayers known as nanodiscs. Winterstein et al. show that they can subsequently insert into planar bilayers, providing a rapid and contamination-free method for functional characterization.

Cited by 35 publications

References 48 publications

A small viral potassium ion channel with an inherent inward rectification

A small viral potassium ion channel with an inherent inward rectification

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

Biomimetic and bioinspired membranes for water purification: A critical review and future directions

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