Structure Of Ion Channels Research Articles

Probing the Molecular Basis of Opposing PUFA Effects on KV7 Channels

The human KV7 ion channel family consists of five members, KV7.1-7.5, distribution of which differs between tissues. As different KV7 subtypes play different roles in different physiological processes, developing pharmacological drugs that differentiate between KV7 subtypes is important in order to limit off-target effects. In pilot experiments, we have observed that free fatty acids, such as the omega-3 fatty acid docosahexaenoic acid, activated all KV7 channels save for KV7.4. In contrast, docosahexaenoic acid induced a slight inhibition of the KV7.4 channel. The aim of this study was to understand the molecular mechanism underlying the difference in observed effects of free fatty acid and their analogues on KV7.4. Using the two-electrode voltage clamp technique to measure potassium currents in Xenopus oocytes expressing hKV7.4 and hKV7.5, we compared the effect of free fatty acids and their analogues on these two KV7 channel subtypes. Our experiments found that neutralisation of the negative charge in the carboxylic head group of docosahexaenoic acid quenched both the inhibition and activation of KV7.4 and KV7.5, respectively, indicating an electrostatic element is involved in the mechanism of action. However, the importance of lipid tail properties differs between the two effects. To determine the contributions of ion channel structure to the observed difference in response, we created KV7.4 mutants in which native amino acids had been substituted for their KV7.5 counterparts. Our experiments found that mutation of residues in a putative fatty acid site either impaired or completely abolished fatty acid-mediated inhibition, but did not result in channel activation when docosahexaenoic acid was applied. Our preliminary studies have identified regions in KV7.4 that contribute to fatty acid-mediated inhibition. Further studies will be performed to fully understand the mechanistic basis for the opposing PUFA effects on KV7.4 and KV7.5.

Structure-Function Relationship and Physiological Roles of Transient Receptor Potential Canonical (TRPC) 4 and 5 Channels.

The study of the structure–function relationship of ion channels has been one of the most challenging goals in contemporary physiology. Revelation of the three-dimensional (3D) structure of ion channels has facilitated our understanding of many of the submolecular mechanisms inside ion channels, such as selective permeability, voltage dependency, agonist binding, and inter-subunit multimerization. Identifying the structure–function relationship of the ion channels is clinically important as well since only such knowledge can imbue potential therapeutics with practical possibilities. In a sense, recent advances in the understanding of the structure–relationship of transient receptor potential canonical (TRPC) channels look promising since human TRPC channels are calcium-permeable, non-selective cation channels expressed in many tissues such as the gastrointestinal (GI) tract, kidney, heart, vasculature, and brain. TRPC channels are known to regulate GI contractility and motility, pulmonary hypertension, right ventricular hypertrophy, podocyte injury, seizure, fear, anxiety-like behavior, and many others. In this article, we tried to elaborate recent findings of Cryo-EM (cryogenic-electron microscopy) based structural information of TRPC 4 and 5 channels and domain-specific functions of the channel, such as G-protein mediated activation mechanism, extracellular modification of the channel, homo/hetero-tetramerization, and pharmacological gating mechanisms.

ImKTx96, a peptide blocker of the Kv1.2 ion channel from the venom of the scorpion Isometrus maculates

Scorpion venom contains diverse bioactive peptides that can recognize and interact with membrane proteins such as ion channels. These natural toxins are believed to be useful tools for exploring the structure and function of ion channels. In this study, we characterized a K+-channel toxin gene, ImKTx96, from the venom gland cDNA library of the scorpion Isometrus maculates. The peptide deduced from the ImKTx96 precursor nucleotide sequence contains a signal peptide of 27 amino acid residues and a mature peptide of 29 residues with three disulfide bridges. Multiple sequence alignment indicated that ImKTx96 is similar with the scorpion toxins that typically target K+-channels. The recombined ImKTx96 peptide (rImKTx96) was expressed in the Escherichia coli system, and purified by GST-affinity chromatography and RP-HPLC. Results from whole-cell patch-clamp experiments revealed that rImKTx96 can inhibit the current of the Kv1.2 ion channel expressed in HEK293 cells. The 3D structure of ImKTx96 was constructed by molecular modeling, and the complex formed by ImKTx96 interacting with the Kv1.2 ion channel was obtained by molecular docking. Based on its structural features and pharmacological functions, ImKTx96 was identified as one member of K+-channel scorpion toxin α-KTx10 group and may be useful as a molecular probe for investigating the structure and function of the Kv1.2 ion channel.

Spatiotemporal Imaging of Water in Operating Voltage-Gated Ion Channels Reveals the Slow Motion of Interfacial Ions

Ion channels are responsible for numerous physiological functions ranging from transport to chemical and electrical signaling. Although static ion channel structure has been studied following a structural biology approach, spatiotemporal investigation of the dynamic molecular mechanisms of operational ion channels has not been achieved experimentally. In particular, the role of water remains elusive. Here, we perform label-free spatiotemporal second harmonic (SH) imaging and capacitance measurements of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by aqueous solution on either side. We observe changes in SH intensity upon channel activation that are traced back to changes in the orientational distribution of water molecules that reorient along the field lines of transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. The time scale of these interfacial changes is influenced by the density of ion channels and is subject to a crowding mechanism. Ion transport along cell membranes is often associated with the propagation of electrical signals in neurons. As our study shows that this process is taking place over seconds, a more complex mechanism is likely responsible for the propagation of neuronal electrical signals than just the millisecond movement of ions.

Genetic Testing in Inherited Arrhythmias: Approach, Limitations, and Challenges

Genetic testing is playing an ever-expanding role in cardiovascular care and is becoming part of the “toolkit” for the cardiovascular clinician. In patients with inherited arrhythmias, genetic testing can confirm a suspected diagnosis, establish a diagnosis in unexplained cases, and help facilitate cascade family screening. Many inherited arrhythmia syndromes are monogenic diseases arising from a single pathogenic variant involved in the structure and function of cardiac ion channels or structural proteins. As such, “arrhythmia gene panels” will often cast a wide net for such heritable diseases. However, challenges may arise when genetic testing results are ambiguous, or when genetic testing results (genotype) and clinical phenotypes do not match. In cases of “genotype-phenotype matching,” genetic results complement the clinical phenotype and genetic testing can be used in diagnosis, family screening, and occasionally prognostication. It becomes more challenging when genetic results are negative or noncontributory and “contradict” the clinical phenotype. “Genotype mismatches” can also occur when genotype-positive patients have no clinical phenotype, or when genetic testing results point towards a completely different disease than the clinical phenotype. We discuss an approach to genetic testing and review the challenges that may arise when interpreting genetic testing results. Genetic testing has opened a wealth of opportunities in the diagnosis, management, and cascade screening of inherited arrhythmia syndromes, but has also opened a “Pandora’s box” of challenges. Genetic results should be interpreted with caution and in a multidisciplinary clinic, with support from genetic counsellors and an expert with a focused interest in cardiovascular genetics.

The contribution of voltage clamp fluorometry to the understanding of channel and transporter mechanisms

Key advances in single particle cryo-EM methods in the past decade have ushered in a resolution revolution in modern biology. The structures of many ion channels and transporters that were previously recalcitrant to crystallography have now been solved. Yet, despite having atomistic models of many complexes, some in multiple conformations, it has been challenging to glean mechanistic insight from these structures. To some extent this reflects our inability to unambiguously assign a given structure to a particular physiological state. One approach that may allow us to bridge this gap between structure and function is voltage clamp fluorometry (VCF). Using this technique, dynamic conformational changes can be measured while simultaneously monitoring the functional state of the channel or transporter. Many of the important papers that have used VCF to probe the gating mechanisms of channels and transporters have been published in the Journal of General Physiology In this review, we provide an overview of the development of VCF and discuss some of the key problems that have been addressed using this approach. We end with a brief discussion of the outlook for this technique in the era of high-resolution structures.

Finding the gates

Structural Biology Hundreds of ion channel structures are available thanks in large part to recent advances in electron microscopy. Insights into channel gating have come from structures of the same channel in different conformational states, supported by biophysical approaches. Gating can occur in hydrophobic regions of the pore even when the diameter is larger than that of a water molecule. Rao et al. simulate the water distribution in nearly 200 representative ion channel structures. Using a machine learning approach, they classify the hydrophobicity–pore radius landscape into two regions, depending on whether water is likely to be excluded or not. This enables them to predict the presence of hydrophobic gates by analysis of the hydrophobicity and pore radius of a single set of coordinates without molecular dynamic simulation of the structure. Proc. Natl. Acad. Sci. U.S.A. 116 , 13989 (2019).

Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel whose α subunit is Kcnma1, and a voltage-dependent calcium channel, Cacna1d. It has also been suggested based on physiological and pharmacological evidence that a voltage-gated K channel is present in the basal membranes of the receptor cells which modulates synaptic transmitter release. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory β subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 using purified mRNA of homogenized electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the Bellono et al. RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent K channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels. The second ortholog is similar to Kv1.5 but has fewer N-terminal amino acids than other species. The sequence for Kv1.5 in the skate is very strongly aligned with the recently reported sequence for potassium channels in the electroreceptors of the cat shark, S. retifer, which also modulate synaptic transmission. The latter channel was designated as Kv1.3 in the initial report, but we suggest that these channels are actually orthologs of each other, and that Kv1.5 is the prevailing designation. We also found a beta subunit sequence (Kcnab2) which may co-assemble with one or both of the voltage gated channels. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of Kcnma1, and found a computer-generated assembly that contained a complete sequence of its β subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 is absent. The evolutionary origin of the newly described K channels and their subunits was studied by alignments with mammalian sequences, including human, and also those in related fish: the whale shark (R. typus), the ghost shark (C.milii), and (S. retifer). There are also orthologous K channels of the lamprey, which has electroreceptors. Tree building and bootstrap programs were used to confirm phylogenetic inferences. Further research should focus on the subcellular locations of these channels, their gating behavior, and the effects of accessory subunits on gating.

NMR Structural Analysis of Isolated Shaker Voltage-Sensing Domain in LPPG Micelles

The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. To probe dynamics with site-specific resolution, we utilized NMR spectroscopy to characterize the VSD derived from Shaker potassium channel in 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPPG) micelles. The backbone dihedral angles predicted based on secondary chemical shifts using torsion angle likeliness obtained from shift (TALOS+) showed that the Shaker-VSD shares many structural features with the homologous Kv1.2/2.1 chimera, including a transition from α-helix to 310 helix in the C-terminal portion of the fourth transmembrane helix. Nevertheless, there are clear differences between the Shaker-VSD and Kv1.2/2.1 chimera in the S2-S3 linker and S3 transmembrane region, where the organization of secondary structure elements in Shaker-VSD appears to more closely resemble the KvAP-VSD. Comparison of microsecond-long molecular dynamics simulations of Kv 1.2-VSD in LPPG micelles and a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer showed that LPPG micelles do not induce significant structural distortion in the isolated voltage sensor. To assess the integrity of the tertiary fold, we directly probed the binding of BrMT analog 2-[2-({[3-(2-amino-ethyl)-6-bromo-1H-indol-2-yl]methoxy}k7methyl)-6-bromo-1H-indol-3-yl]ethan-1-amine (BrET), a gating modifier toxin, and identified the location of the putative binding site. Our results suggest that the Shaker-VSD in LPPG micelles is in a native-like fold and is likely to provide valuable insights into the dynamics of voltage-gating and its regulation.

A heuristic derived from analysis of the ion channel structural proteome permits the rapid identification of hydrophobic gates

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined; however, these newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore, and it is now known that more accurate annotation of their conductive state requires additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavor liquid-phase water, leading to local dewetting, which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore dewetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but also may facilitate the design of novel nanopores controlled by hydrophobic gates.

A bionic stretchable nanogenerator for underwater sensing and energy harvesting

Soft wearable electronics for underwater applications are of interest, but depend on the development of a waterproof, long-term sustainable power source. In this work, we report a bionic stretchable nanogenerator for underwater energy harvesting that mimics the structure of ion channels on the cytomembrane of electrocyte in an electric eel. Combining the effects of triboelectrification caused by flowing liquid and principles of electrostatic induction, the bionic stretchable nanogenerator can harvest mechanical energy from human motion underwater and output an open-circuit voltage over 10 V. Underwater applications of a bionic stretchable nanogenerator have also been demonstrated, such as human body multi-position motion monitoring and an undersea rescue system. The advantages of excellent flexibility, stretchability, outstanding tensile fatigue resistance (over 50,000 times) and underwater performance make the bionic stretchable nanogenerator a promising sustainable power source for the soft wearable electronics used underwater.

CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores

The control of ion channel permeation requires the modulation of energetic barriers or “gates” within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behavior of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit “hydrophobic gating” to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.

P2Y1 Purinergic Receptor Modulate Axon Initial Segment Initial Development.

Morphological and functional polarization of neurons depends on the generation and maintenance of the axon initial segment (AIS). This axonal domain maintains axonal properties but is also the place where the action potential (AP) is generated. All these functions require the AIS, a complex structure that is not fully understood. An integrated structure of voltage-gated ion channels, specific cytoskeleton architecture, as well as, scaffold proteins contributes to these functions. Among them, ankyrinG plays a crucial role to maintain ion channels and membrane proteins. However, it is still elusive how the AIS performs its complex structural and functional regulation. Recent studies reveal that AIS is dynamically regulated in molecular composition, length and location in response to neuronal activity. Some mechanisms acting on AIS plasticity have been uncovered recently, including Ca2+, calpain or calmodulin-mediated modulation, as well as post-translational modifications of cytoskeleton proteins and actin-associated proteins. Neurons are able to respond to different kind of physiological and pathological stimuli from development to maturity by adapting their AIS composition, position and length. This raises the question of which are the neuronal receptors that contribute to the modulation of AIS plasticity. Previous studies have shown that purinergic receptor P2X7 activation is detrimental to AIS maintenance. During initial axonal elongation, P2X7 is coordinated with P2Y1, another purinergic receptor that is essential for proper axon elongation. In this study, we focus on the role of P2Y1 receptor on AIS development and maintenance. Our results show that P2Y1 receptor activity and expression are necessary during AIS initial development, while has no role once AIS maturity is achieved. P2Y1 inhibition or suppression results in a decrease in ankyrinG, βIV-spectrin and voltage-gated sodium channels accumulation that can be rescued by actin stabilization or the modulation of actin-binding proteins at the AIS. Moreover, P2X7 or calpain inhibition also rescues ankyrinG decrease. Hence, a dynamic balance of P2Y1 and P2X7 receptors expression and function during AIS assembly and maturation may represent a fine regulatory mechanism in response to physiological or pathological extracellular purines concentration.

The Role of Toxins in the Pursuit for Novel Analgesics.

Chronic pain is a major medical issue which reduces the quality of life of millions and inflicts a significant burden on health authorities worldwide. Currently, management of chronic pain includes first-line pharmacological therapies that are inadequately effective, as in just a portion of patients pain relief is obtained. Furthermore, most analgesics in use produce severe or intolerable adverse effects that impose dose restrictions and reduce compliance. As the majority of analgesic agents act on the central nervous system (CNS), it is possible that blocking pain at its source by targeting nociceptors would prove more efficient with minimal CNS-related side effects. The development of such analgesics requires the identification of appropriate molecular targets and thorough understanding of their structural and functional features. To this end, plant and animal toxins can be employed as they affect ion channels with high potency and selectivity. Moreover, elucidation of the toxin-bound ion channel structure could generate pharmacophores for rational drug design while favorable safety and analgesic profiles could highlight toxins as leads or even as valuable therapeutic compounds themselves. Here, we discuss the use of plant and animal toxins in the characterization of peripherally expressed ion channels which are implicated in pain.

Effect of norepinephrine treatment on Haemonchus contortus and its excretory products.

Haemonchus contortus is a highly pathogenic gastrointestinal nematode of small ruminant animals. In modern intensive farming, livestock often suffer from different types of stress. However, whether host stress hormones influence H. contortus infection is largely unknown. Therefore, we treated H. contortus with norepinephrine (NE) and analyzed the changes in its excretory/secretory products (ESPs). Label-free quantitative proteomic analysis was used to identify differences in body proteins and ESPs between the control and NE-treated groups. We also investigated the changes in ESP action by analyzing cytokine secretion and goat peripheral blood mononuclear cell (PBMC) proliferation after incubation with ESPs secreted by NE-treated H. contortus. Thirty-two proteins in the body samples and 137 in the ESPs were differentially expressed between the groups. Gene ontology (GO) annotation showed that the functions of these different proteins might be involved in energy metabolism, protein metabolism, lipid metabolism, redox homeostasis, ion channel, and cell structure. NE treatment caused oxidative stress in H. contortus and changed the expression levels of some immunogenic proteins, such as the 15-kDa ESP. Meanwhile, the ESPs secreted by NE-treated H. contortus significantly decreased PBMC proliferation and the interleukin (IL)-2, IL-4, and interferon-gamma contents. Thus, NE treatment significantly affected the H. contortus body and ESP expression, and changes in the ESPs influenced PBMC function. The results reveal a relationship between host hormones and parasites and provide new clues to explain some of the variation in individual responses to infection.

Role of the Cytoplasmatic M3-M4 Loop for the Homopentameric Assembly of a Chimeric Nicotinic Alpha 7 Receptor

It is long known that the nicotinic acetylcholine receptor α7 (CHRNA7R) expresses weakly as a functional acetylcholine-gated ion channel in many cells. Using the blue native PAGE (BN-PAGE) technique for visualization of oligomers, we have previously shown that singly expressed CHRNA7R subunits assemble in X. laevis oocytes to lower order homooligomers, mostly tetramers; only a small fraction of the synthesized CHRNA7R subunits acquire the fully assembled homopentameric state, in which they can escape the endoplasmic reticulum to be routed to the plasma membrane (Nicke et al, FEBS Lett 575, 52-58. 2004). Here we show that a previously described chimeric receptor (Eiselé et al, Nature 366, 479-483, 1993) comprising the extracellular domain of the CHRNA7R fused to the transmembrane spanning and intracellular domains of the 5HT3A receptor (designated rCHRNA7V2015HT3AR chimera) assembled as efficiently to homopentamers as the 5HT3AR. The large cytoplasmatic M3-M4 loop of the 5HT3AR has previously been identified to represent an oligomerization domain that assembles into stable homopentamers when expressed as a soluble protein without flanking transmembrane helices (Pandhare et al, Sci. Rep. 1-9, 2016). To define the 5HT3AR sequence needed for efficient homopentameric assembly, we systematically varied the sequence encoding the M3-M4 loop of the rCHRNA7V2015HT3AR chimera from 5HT3A to CHRNA7R. We analyzed the resulting chimeras by BN-PAGE, SDS-PAGE and two-electrode voltage-clamp electrophysiology. We found that assembly of functional homopentameric receptors was largely abolished when the CHRNA7R sequence was present into the proximal half of the M3-M4 loop. By a detailed mapping of the first half of the M3-M4 loop we identified a sequence of six amino acids being indispensable for efficient subunit assembly. We will interpret these results on the basis of known 3D structures of ligand-gated ion channels.

Functional Annotation of Ion Channel Structures: Predicting Pore Solvation States Based on Local Radius and Hydrophobicity

Functional Annotation of Ion Channel Structures: Predicting Pore Solvation States Based on Local Radius and Hydrophobicity

Broken Symmetry in the Human BK Channel

Voltage-gated and ligand-modulated ion channels play critical roles in excitable cells. To understand the interplay among voltage-sensing, ligand-binding and channel opening, the structures of ion channels in various functional states need to be determined. Here, the “random spherically constrained” (RSC) single-particle cryo-EM method was employed to study the human large conductance voltage- and calcium-activated potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK structure has been determined at 3.5 A resolution in the absence of Ca2+. Instead of the common four-fold symmetry observed in ligand-modulated ion channels, a two-fold symmetry was observed in hBK. Two opposing subunits in the Ca2+ sensing gating ring rotate around the center of each subunit, which results in the movement of the assembly and flexible interfaces and Ca2+ binding sites. Despite the significant movement, the local conformation of the assembly interfaces and Ca2+ binding sites remains the same among the four subunits.

Applications for Mass Spectrometry in theStudy of Ion Channel Structure and Function.

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca2+, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca2+ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

Exploiting the Diversity of Ion Channels: Modulation of Ion Channels for Therapeutic Indications.

Ion channels are macromolecular proteins that form water-filled pores in cell membranes and they are critical for a variety of physiological and pharmacological functions. Dysfunctional ion channels can cause diseases known as channelopathies. Ion channels are encoded by approximately 400 genes, representing the second largest class of proven drug targets for therapeutic areas including neuropsychiatric disorders, cardiovascular and metabolic diseases, immunological diseases, nephrological diseases, gastrointestinal diseases, pulmonary/respiratory diseases, and many cancers. With more ion channel structures are being solved and functional robust assays are being developed, there are tremendous opportunities for identifying specific modulators targeting ion channels for new therapy.