Engineering Of Ion Channels Research Articles

Disorders associated with Ion Channels

Muscular wasting, a lack of muscle tone, or sporadic muscle paralysis are the most common symptoms of ion channel diseases, which are linked to defects in the proteins known as ion channels. Ion channels are diverse and differ with respect to how they open and close (gating) and their ionic conductance and selectivity (permeation). The development of novel molecules to modulate their activity, fundamental understanding of ion channel structure-function mechanisms, their physiological functions, how their dysfunction results in disease, their use as biosensors, and their utility as biosensors are important and active research frontiers. With a particular emphasis on voltage-gated ion channels, ion-channel engineering methods that have been used to investigate these facets of ion channel function are x-rayed.

Ion Channel Engineering in Super Thick Cathodes toward High-Energy-Density Li–S Batteries

Ion Channel Engineering in Super Thick Cathodes toward High-Energy-Density Li–S Batteries

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

SummaryLigand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

Ion channel engineering using protein trans-splicing.

Conventional site-directed mutagenesis and genetic code expansion approaches have been instrumental in providing detailed functional and pharmacological insight into membrane proteins such as ion channels. Recently, this has increasingly been complemented by semi-synthetic strategies, in which part of the protein is generated synthetically. This means a vast range of chemical modifications, including non-canonical amino acids (ncAA), backbone modifications, chemical handles, fluorescent or spectroscopic labels and any combination of these can be incorporated. Among these approaches, protein trans-splicing (PTS) is particularly promising for protein reconstitution in live cells. It relies on one or more split inteins, which can spontaneously and covalently link flanking peptide or protein sequences. Here, we describe the use of PTS and its variant tandem PTS (tPTS) in semi-synthesis of ion channels in Xenopus laevis oocytes to incorporate ncAAs, post-translational modifications or metabolically stable mimics thereof. This strategy has the potential to expand the type and number of modifications in ion channel research.

Ion-Channels: Goals for Function-Oriented Synthesis

Ion channels provide a conductance pathway for the passive transport of ions across membranes. These functional molecules perform key tasks in biological systems such as neuronal signaling, muscular control, and sensing. Recently, function-oriented synthesis researchers began to focus on ion channels with the goal of modifying the function of existing ion channels (ion selectivity, gating) or creating new channels with novel functions. Both approaches, ion channel engineering and de novo design, have involved synthetic chemists, biochemists, structural biologists, and neurochemists. Researchers characterize the function of ion channels by measuring their conductance in samples of biological membranes (patch clamp) or artificial membranes (planar lipid bilayers). At the single molecule level, these measurements require special attention to the purity of the sample, a challenge that synthetic chemists should be aware of. Ideally, researchers study the function of channels while also acquiring structural data (X-ray, NMR) to understand and predict how synthetic modifications alter channel function. Long-term oriented researchers would like to apply synthetic ion channels to single molecule sensing and to implantat these synthetic systems in living organisms as tools or for the treatment of channelopathies. In this Account, we discuss our own work on synthetic ion channels and explain the shift of our research focus from a de novo design of oligo-THFs and oligo-THF-amino acids to ion channel engineering. We introduce details about two biological lead structures for ion channel engineering: the gramicidin β(6,3) helix as an example of a channel with a narrow ion conductance pathway and the outer membrane porins (OmpF, OmpG) with their open β-barrel structure. The increase and the reversal of ion selectivity of these systems and the hydrophobic match/mismatch of the channel with the phospholipid bilayer are of particular interest. For engineering ion channels, we need to supplement the single-point attachment of a synthetic modulator with the synthesis of a more challenging two-point attachment. The successful function-oriented synthesis of ion channels will require interdisciplinary efforts that include new electrophysiology techniques, efficient synthesis (peptide/protein/organic), and good structural analysis.

ChemInform Abstract: Strategies and Perspectives in Ion‐Channel Engineering

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Strategies and Perspectives in Ion‐Channel Engineering

Membranes form natural barriers that need to be permeable to diverse matter like ions and substrates. This permeability is controlled by ion-channel proteins, which have attracted great interest for pharmaceutical applications. Ion-channel engineering (ICE) modifies biological ion channels by chemical/biological synthetis means. The goal is to obtain ion channels with modified or novel functionality. Three functional strategies exist. The first is the manipulation of the wider pores with robust β-barrel structures, such as those of α-hemolysin and porins. The second engineering approach focuses on the modification of narrow (mostly α-helical) pores to understand selectivity and modes of action. A third functional approach addresses channel gating by (photo)triggering the biological receptor that controls the channel. Several synthetis strategies have been developed and successfully utilized for the synthetic modification of biological ion-channels: the S-alkylation of specifically introduced Cys, protein semisynthesis by native chemical ligation, protein semisynthesis by protein trans-splicing, as well as nonsense-suppression methods. Structural studies (X-ray crystallography, NMR spectroscopy) are necessary to support the functional studies and to afford predictable engineering. The reprogramming and re-engineering of channels can be used for sensing applications, treatment of channelopathies, chemical neurobiology, and providing novel lead compounds for targeting ion channels.

Ion-channel engineering

The channel-mediated transport of ions through a membrane is of great importance in biological systems and holds a considerable potential for sensing applications. Ion-channel engineering follows two approaches. The first is the engineering of biological channels with the goal to control existing functionality. Currently, this area benefits from progress in structural information of ion channels, new methods in protein engineering (protein semi-synthesis), and the inclusion of chemical synthesis to obtain hybrid ion channels. The second approach in ion channel engineering is the de novo design. Here, progress emerges from self-assembly concepts to prepare β-barrel and π-barrel pores. This review focuses on contributions published in the year 2007 with inclusion of selected work of the time span 2005–2006.

Chemical Synthesis Approaches to the Engineering of Ion Channels

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Conversion of a Porin-like Peptide Channel into a Gramicidin-like Channel by Glycine to D-alanine Substitutions

The β-barrel and β-helix formation, as in porins and gramicidin, respectively, represent two distinct mechanisms for ion channel formation by β-sheet proteins in membranes. The design of β-barrel proteins is difficult due to incomplete understanding of the basic principles of folding. The design of gramicidin-like β-helix relies on an alternating pattern of L- and D-amino acid sequences. Recently we noticed that a short β-sheet peptide (xSxG)6, can form porin-like channels via self-association in membranes. Here, we proposed that glycine to D-alanine substitutions of the N-formyl-(xSxG)6 would transform the porin-like channel into a gramicidin-like β12-helical channel. The requirement of an N-formyl group for channel activity, impermeability to cations with a diameter >4Å, high monovalent cation selectivity, and the absence of either voltage gating or subconductance states upon D-alanine substitution support the idea of a gramicidin-like channel. Moreover, the circular dichroism spectrum in membranes is different, indicating a change in regular β-sheet backbone structure. The conversion of a complex porin-like channel into a gramicidin-like channel provides a link between two different mechanisms of β-sheet channel formation in membranes and emphasizes the importance of glycine and D-amino acid residues in protein folding and function and in the engineering of ion channels.

Chemical Synthesis Approaches to the Engineering of Ion Channels

Chemoselective ligation strategies have previously provided synthetic access to water-soluble proteins with novel properties, and more recently these strategies have been used to prepare ion channels. Examples of ion channels prepared by total chemical synthesis include bacterial mechanosensitive channels, and viral ion channels. Chemical protein synthesis allows for the generation of ion channel proteins with both native, and engineered structural or conductance properties.