Ion Channel Research Research Articles

Using Nanoscale Passports To Understand and Unlock Ion Channels as Gatekeepers of the Cell.

Natural ion channels are proteins embedded in the cell membrane that control many aspects of cell and human physiology by acting as gatekeepers, regulating the flow of ions in and out of cells. Advances in nanotechnology have influenced the methods for studying ion channels in vitro, as well as ways to unlock the delivery of therapeutics by modulating them in vivo. This review provides an overview of nanotechnology-enabled approaches for ion channel research with a focus on the synthesis and applications of synthetic ion channels. Further, the uses of nanotechnology for therapeutic applications are critically analyzed. Finally, we provide an outlook on the opportunities and challenges at the intersection of nanotechnology and ion channels. This work highlights the key role of nanoscale interactions in the operation and modulation of ion channels, which may prompt insights into nanotechnology-enabled mechanisms to study and exploit these systems in the near future.

Research Progress on Plant Shaker K+ Channels.

Plant growth and development are driven by intricate processes, with the cell membrane serving as a crucial interface between cells and their external environment. Maintaining balance and signal transduction across the cell membrane is essential for cellular stability and a host of life processes. Ion channels play a critical role in regulating intracellular ion concentrations and potentials. Among these, K+ channels on plant cell membranes are of paramount importance. The research of Shaker K+ channels has become a paradigm in the study of plant ion channels. This study offers a comprehensive overview of advancements in Shaker K+ channels, including insights into protein structure, function, regulatory mechanisms, and research techniques. Investigating Shaker K+ channels has enhanced our understanding of the regulatory mechanisms governing ion absorption and transport in plant cells. This knowledge offers invaluable guidance for enhancing crop yields and improving resistance to environmental stressors. Moreover, an extensive review of research methodologies in Shaker K+ channel studies provides essential reference solutions for researchers, promoting further advancements in ion channel research.

Structural modeling of ion channels using AlphaFold2, RoseTTAFold2, and ESMFold

ABSTRACT Ion channels play key roles in human physiology and are important targets in drug discovery. The atomic-scale structures of ion channels provide invaluable insights into a fundamental understanding of the molecular mechanisms of channel gating and modulation. Recent breakthroughs in deep learning-based computational methods, such as AlphaFold, RoseTTAFold, and ESMFold have transformed research in protein structure prediction and design. We review the application of AlphaFold, RoseTTAFold, and ESMFold to structural modeling of ion channels using representative voltage-gated ion channels, including human voltage-gated sodium (NaV) channel - NaV1.8, human voltage-gated calcium (CaV) channel – CaV1.1, and human voltage-gated potassium (KV) channel – KV1.3. We compared AlphaFold, RoseTTAFold, and ESMFold structural models of NaV1.8, CaV1.1, and KV1.3 with corresponding cryo-EM structures to assess details of their similarities and differences. Our findings shed light on the strengths and limitations of the current state-of-the-art deep learning-based computational methods for modeling ion channel structures, offering valuable insights to guide their future applications for ion channel research.

Label-free optical detection of calcium ion influx in cell-derived nanovesicles using a conical Au/PDMS biosensor.

Ion channels, which are key to physiological regulation and drug discovery, control ion flux across membranes, and their dysregulation leads to various diseases. Ca2+ monitoring is crucial for cellular signaling when performing Ca-based assays in ion channel research; these assays are widely utilized in both academic and pharmaceutical contexts for drug screening and pharmacological profiling. However, existing detection methods are limited by slow detection speeds, low throughput, complex processes, and low analyte viability. In this study, we developed a label-free optical biosensing method using a conical Au/polydimethylsiloxane platform tailored to detect Ca2+ influx in A549-originated nanovesicles facilitated by the transient receptor potential ankyrin 1 (TRPA1) channel. Nanovesicles expressing cellular signaling components mimic TRPA1 signal transduction in cell membranes and improve analyte viability. The conical Au/polydimethylsiloxane sensor converted Ca2+ influx events induced by specific agonist exposure into noticeable changes in relative transmittance under visible light. The optical transmittance change accompanying Ca2+ influx resulted in an enhanced sensing response, high accuracy and reliability, and rapid detection (∼5 s) without immobilization or ligand treatments. In the underlying sensing mechanism, morphological variations in nanovesicles, which depend on Ca2+ influx, induce a considerable light scattering change at an interface between the nanovesicle and Au, revealed by optical simulation. This study provides a foundation for developing biosensors based on light-matter interactions. These sensors are simple and cost-effective with superior performance and diverse functionality.

News and views on ion channels in cancer: is cancer a channelopathy?

Ion channels are key signaling proteins found throughout the body; they are critical in many, wide-ranging physiological processes, from gene expression, sensory perception and processing to the cardiac action potential. When ion channel activity goes awry, for example, via mutation, damage or disrupted homeostasis, the outcome can result in causation, development and/or maintenance of disease. Ion channel dependent diseases have been dubbed channelopathies. Recent studies on the role of ion channels in cancer biology suggest that cancer is one such channelopathy. Many ion channels have now been implicated in the cellular processes that are affected in a multitude of cancers. In the last two decades, the field of ion channel and cancer research has been growing exponentially: a combination of developments in molecular biology, genetics, electrophysiology and automation have driven an explosion in our capabilities to interrogate ion channel pathways; how, why and where they go wrong and therapeutic interventions to correct their pathophysiology in cancer. A review of this vast and rapidly developing field would require a titanic tome to merely dimple the surface of research that has ballooned recently. In lieu of that huge undertaking—for the benefit of both authors and readers - this review discusses select examples of primary, applied and clinical research, aiming to shine a light on some of the more innovative and novel findings that this exciting field is excavating.

5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 μM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 μM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 μM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Rapid Pacing Decreases L-type Ca2+ Current and Alters Cacna1c Isogene Expression in Primary Cultured Rat Left Ventricular Myocytes

The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaL directly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+ channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaL in different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaL density by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of total cacna1c and of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaL density by 30%, mildly slowed ICaL inactivation and shifted steady-state inactivation to more negative potentials. Total cacna1c mRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaL density and cacna1c mRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes.Graphical

Historical Perspective of the Characterization of Conotoxins Targeting Voltage-Gated Sodium Channels.

Marine toxins have potent actions on diverse sodium ion channels regulated by transmembrane voltage (voltage-gated ion channels) or by neurotransmitters (nicotinic acetylcholine receptor channels). Studies of these toxins have focused on varied aspects of venom peptides ranging from evolutionary relationships of predator and prey, biological actions on excitable tissues, potential application as pharmacological intervention in disease therapy, and as part of multiple experimental approaches towards an understanding of the atomistic characterization of ion channel structure. This review examines the historical perspective of the study of conotoxin peptides active on sodium channels gated by transmembrane voltage, which has led to recent advances in ion channel research made possible with the exploitation of the diversity of these marine toxins.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

The contribution of ion channels to shaping macrophage behaviour.

The expanding roles of macrophages in physiological and pathophysiological mechanisms now include normal tissue homeostasis, tissue repair and regeneration, including neuronal tissue; initiation, progression, and resolution of the inflammatory response and a diverse array of anti-microbial activities. Two hallmarks of macrophage activity which appear to be fundamental to their diverse cellular functionalities are cellular plasticity and phenotypic heterogeneity. Macrophage plasticity allows these cells to take on a broad spectrum of differing cellular phenotypes in response to local and possibly previous encountered environmental signals. Cellular plasticity also contributes to tissue- and stimulus-dependent macrophage heterogeneity, which manifests itself as different macrophage phenotypes being found at different tissue locations and/or after different cell stimuli. Together, plasticity and heterogeneity align macrophage phenotypes to their required local cellular functions and prevent inappropriate activation of the cell, which could lead to pathology. To execute the appropriate function, which must be regulated at the qualitative, quantitative, spatial and temporal levels, macrophages constantly monitor intracellular and extracellular parameters to initiate and control the appropriate cell signaling cascades. The sensors and signaling mechanisms which control macrophages are the focus of a considerable amount of research. Ion channels regulate the flow of ions between cellular membranes and are critical to cell signaling mechanisms in a variety of cellular functions. It is therefore surprising that the role of ion channels in the macrophage biology has been relatively overlooked. In this review we provide a summary of ion channel research in macrophages. We begin by giving a narrative-based explanation of the membrane potential and its importance in cell biology. We then report on research implicating different ion channel families in macrophage functions. Finally, we highlight some areas of ion channel research in macrophages which need to be addressed, future possible developments in this field and therapeutic potential.

In-situ and amplification-free imaging of hERG ion channels at single-cell level using a unique core-molecule-shell-secondary antibody SERS nanoprobe

Research on ion channels and their monoclonal antibodies plays a critical role in drug development and disease diagnosis. The current ion channel researches are often not conducted in the microenvironment for cells survival, which restricts the mechanism study of the links between the cell structure and the ion channel function. In this work, we synthesized gold core-4-mercaptobenzonitrile-sliver shell-goat anti-rabbit immunoglobulin G (Au@4-MBN@Ag@IgG) nanoparticles as surface-enhanced Raman scattering (SERS) nanoprobes for investigating the human ether-a-go-go related gene (hERG) potassium ion channel in cell membranes. This is the first attempt to study ion channels using SERS. Due to the unique core-molecule-shell structure and the silver shell of nanoprobes, strong and stable SERS signal was obtained. With the help of antibodies, the Au@4-MBN@Ag@IgG nanoprobes were captured by hERG antibodies and then bonded with hERG ion channels based on the sandwich immune response. The reporter molecule, 4-MBN, displayed a strong and sharp characteristic peak at 2233 cm-1 in the Raman silent region. The intensity of this peak denoted the concentration of antibodies and the expression of ion channel proteins. We successfully applied this amplification-free method for in-situ imaging the distribution of the hERG ion channel on the transfected HEK293 cell surface at the single-cell level. This hERG ion channel profiling strategy promises a maneuverable tool for ion channel research.

An efficient and scalable data analysis solution for automated electrophysiology platforms.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

Sodium Channels and Local Anesthetics—Old Friends With New Perspectives

The long history of local anesthetics (LAs) starts out in the late 19th century when the content of coca plant leaves was discovered to alleviate pain. Soon after, cocaine was established and headed off to an infamous career as a substance causing addiction. Today, LAs and related substances—in modified form—are indispensable in our clinical everyday life for pain relief during and after minor and major surgery, and dental practices. In this review, we elucidate on the interaction of modern LAs with their main target, the voltage-gated sodium channel (Navs), in the light of the recently published channel structures. Knowledge of the 3D interaction sites of the drug with the protein will allow to mechanistically substantiate the comprehensive data available on LA gating modification. In the 1970s it was suggested that LAs can enter the channel pore from the lipid phase, which was quite prospective at that time. Today we know from cryo-electron microscopy structures and mutagenesis experiments, that indeed Navs have side fenestrations facing the membrane, which are likely the entrance for LAs to induce tonic block. In this review, we will focus on the effects of LA binding on fast inactivation and use-dependent inhibition in the light of the proposed new allosteric mechanism of fast inactivation. We will elaborate on subtype and species specificity and provide insights into modelling approaches that will help identify the exact molecular binding orientation, access pathways and pharmacokinetics. With this comprehensive overview, we will provide new perspectives in the use of the drug, both clinically and as a tool for basic ion channel research.

Controlling ion channel function with renewable recombinant antibodies.

Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.

An inspiration to the studies on mechanisms of acupuncture and moxibustion action derived from 2021 Nobel Prize in Physiology or Medicine

Abstract The editorial board of Acupuncture and Herbal Medicine recently convened an academic seminar with a focus on studies regarding the mechanisms mediating acupuncture efficacy and moxibustion action inspired by the 2021 Nobel Prize in Physiology or Medicine. Specifically, Professor Bailong Xiao introduced the Nobel Prize for research on the mechanically activated Piezo ion channel, evaluating the structure of the Piezo channel and its physiological and pathological functions, and proposed a possible role for the Piezo channel in acupuncture mechanical stimulation. Professor Michael Xi Zhu introduced the discovery of the transient receptor potential (TRP) family, reporting that the therapeutic effects of Chinese medicine and acupuncture may be achieved via the TRP family, and that information regarding associations between the meridian and lymphatic systems may have important research and medical value. In addition, Professor Tianle Xu reviewed the history of ion channel research, particularly the physiological and pharmacological effects of non-classical ion channels (eg, the acid sensing ion channel family) and pointed out that the characterization and neural circuits of acupuncture deqi manipulation are important for elucidating the mechanisms of acupuncture actions. Professor Yongming Li similarly proposed that the 2021 Nobel Prize may open the door to disclosing the histological basis of acupuncture and moxibustion and analyzing the main scientific concerns regarding the clinical translation of acupuncture and moxibustion from basic to translational research. Finally, Professor Yi Guo summarized the study progress of the acupoint microenvironment induced by acupuncture over the course of nearly 30 years and put forward the hypothesis that acupuncture may initiate the physical-chemical coupling network by activating ion channel receptors in acupoints via physical and mechanical stimulation. Therefore, we conclude that a primary achievement of the 2021 Nobel Prize in Physiology or Medicine is in helping interpret how acupuncture and moxibustion adjust homeostasis (ie, by activating mechanical and thermal sensation), which is conducive to validating and promoting the clinical efficacy of acupuncture modalities.

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin.

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (K d for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Ion Channels Orchestrate Pancreatic Ductal Adenocarcinoma Progression and Therapy.

Pancreatic ductal adenocarcinoma is a devastating disease with a dismal prognosis. Therapeutic interventions are largely ineffective. A better understanding of the pathophysiology is required. Ion channels contribute substantially to the “hallmarks of cancer.” Their expression is dysregulated in cancer, and they are “misused” to drive cancer progression, but the underlying mechanisms are unclear. Ion channels are located in the cell membrane at the interface between the intracellular and extracellular space. They sense and modify the tumor microenvironment which in itself is a driver of PDAC aggressiveness. Ion channels detect, for example, locally altered proton and electrolyte concentrations or mechanical stimuli and transduce signals triggered by these microenvironmental cues through association with intracellular signaling cascades. While these concepts have been firmly established for other cancers, evidence has emerged only recently that ion channels are drivers of PDAC aggressiveness. Particularly, they appear to contribute to two of the characteristic PDAC features: the massive fibrosis of the tumor stroma (desmoplasia) and the efficient immune evasion. Our critical review of the literature clearly shows that there is still a remarkable lack of knowledge with respect to the contribution of ion channels to these two typical PDAC properties. Yet, we can draw parallels from ion channel research in other fibrotic and inflammatory diseases. Evidence is accumulating that pancreatic stellate cells express the same “profibrotic” ion channels. Similarly, it is at least in part known which major ion channels are expressed in those innate and adaptive immune cells that populate the PDAC microenvironment. We explore potential therapeutic avenues derived thereof. Since drugs targeting PDAC-relevant ion channels are already in clinical use, we propose to repurpose those in PDAC. The quest for ion channel targets is both motivated and complicated by the fact that some of the relevant channels, for example, KCa3.1, are functionally expressed in the cancer, stroma, and immune cells. Only in vivo studies will reveal which arm of the balance we should put our weights on when developing channel-targeting PDAC therapies. The time is up to explore the efficacy of ion channel targeting in (transgenic) murine PDAC models before launching clinical trials with repurposed drugs.

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.

Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms.

Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.

Production of recombinant venom peptides as tools for ion channel research.

Animal venom is a rich source for peptide toxins that bind and modulate the function of ion channels. Owing to their ability to bind receptor sites on the channel protein with high affinity and specificity, peptide neurotoxins have become an indispensable tool for ion channel research. Recent breakthroughs in structural biology and advances in computer simulations of biomolecules have sparked a new interest in animal toxins as probes of channel protein structure and function. Here, we focus on methods used to produce animal toxins for research purposes using recombinant expression. The specific challenges associated with heterologous production of venom peptides are discussed, and several methods targeting these issues are presented with an emphasis on E. coli based systems. An efficient protocol for the bacterial expression, folding, and purification of recombinant venom peptides is described.