After ligand binding, many ion channels undergo rearrangements at the voltage sensor domain (VSD) that often modulate their gating activity with important physiological repercussions. Since the VSD is dynamic, it is interesting to establish a correlation between the potential mobility of this element in terms of its intrinsic flexibility and its ability to accommodate several ligands by induced-fit mechanisms. We presume that these associations are not causal since the flexibility of the VSD could have an important impact on the ligand coupling event. Many significantly flexible ion channels show a general architecture and composition compatible with important conformational changes and capable of accommodating chemically diverse agonists. In this contribution, the structural bases of this subtle and probably unexpected relationship between the VSD flexibility and its influence during the dynamic coupling of the ligand are exposed. Thus, given its physiological relevance, the study of ion channel malfunction can be associated with ligand accommodation events to the VSD, which could depend on its local flexibility. This could contribute to a better understanding of the molecular bases of a variety of physiological disorders. In consequence, considering these effects during the protein/ligand interaction could be determinant to the rational design of novel drugs.
Mitochondria play a pivotal role in cardioprotection. The major cardioprotective mechanism is ischemic preconditioning (IpreC), through which short periods of ischemia protect a subsequent prolonged acute ischemic episode. Mitochondria channels, particularly the potassium channels (mitoK) such as ATP-dependent and calcium-activated potassium channels, have been suggested as trigger or end effectors in IpreC. Activators of mitoK are promising therapeutic agents for the treatment of the myocardial injury due to ischemic episodes. In this chapter, we are summarizing our current knowledge on the physiology function of different mitochondrial channels with a focus on the potassium channels and their mechanism in cardioprotection. Furthermore, the currently under development therapy by targeting the mitochondrial channels for the treatment of heart failure are also discussed.
The vast majority of morbidity and mortality in cystic fibrosis (CF) patients is due to lung disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). CFTR is a PKA-regulated anion channel localized in the apical membrane of bronchial epithelia where it controls salt and fluid regulation to facilitate bacteria clearance. There are over 1000 disease-causing mutations in the gene encoding CFTR and, depending on the type of mutation, the cell surface density and/or functional activity of CFTR in the apical membrane is reduced. A therapeutic strategy for the treatment of lung disease in CF patients is to use pharmacological agents that increase mutant CFTR-mediated anion secretion. High-throughput screening strategies have identified multiple chemotypes that increase mutant CFTR-mediated anion secretion. These chemotypes can be grouped into two classes based on their mode of action. The first class is known as CFTR correctors because they correct the processing and trafficking of CFTR to increases its cell surface density. The second class is known as CFTR potentiators as they potentiate the amount of anion secretion through CFTR at the cell surface. In vivo analysis of CFTR activity in CF patients indicates that it is correlated with the severity of lung disease and supports the hypothesis that CFTR modulators that restore mutant CFTR activity to >10% of wild-type-CFTR would improve lung function. The use of high-throughput screening and medicinal chemistry optimization to improve the efficacy, potency, and pharmaceutical properties of the multiple potentiator or corrector scaffolds identified to date offers a promising approach for the treatment of CF by directly targeting the root cause of the disease.
Cell proliferation and cell death are two counterparts in sharing the responsibility for maintaining normal body function, and the delicate balance between the two coordinates developmental morphogenesis, cell homeostasis, and tissue modeling in organisms. Abnormally enhanced proliferation and/or impaired cell death often cause loss of control of cell growth leading to tumorigenesis or cancer formation. Several fundamental steps need to be fulfilled at the cellular level for tumorigenesis and these steps can be roughly viewed as characteristic alterations of some physicochemical processes: cell volume, intracellular Ca2+, and intracellular pH. Evidence has rapidly emerged indicating a pivotal role of K+ channels in controlling these fundamental biological processes and a deregulated expression of potassium channel protein-coding genes, as well as malfunction of K+ channels as an important step in the development and progression of cancers. Herein, the role of K+ channels in cancer progression will be introduced by presenting the data obtained over the past 25 years, beginning with the evidence for K+ channels as cancer markers, followed by the data linking K+ channels to neoplastic growth and cancer metastasis. The potential of targeting K+ channels for cancer therapy will then be discussed by outlining the promising approaches and strategies including inhibition of K+ channel activities using pharmacological agents and downregulation of K+ channel expression using various nucleic acids (siRNA, decoy ODN and miRNA). Some unanswered questions and unsolved problems with respect to K+ channels and cancer are discussed in the final section.
Blockers of voltage-gated sodium channels have several therapeutic uses, including use as anticonvulsants, antiarrhythmics, and analgesics. However, voltage-gated sodium channels are challenging drug targets, and most of the clinically used drugs were found before their sodium channel blocking mechanism was known. Sodium channels are a family of ten homologous subtypes, and family members are expressed differentially throughout the nervous system and in cardiac and skeletal muscle tissue. They exist in closed, open, and inactivated conformational states, and selective interactions with one or more of these states differentiates therapeutically useful drugs from neurotoxins. Therefore, assays used in drug discovery need to be sensitive to these mechanisms of action and preferably able to distinguish between drug interactions with different conformational states. Electrophysiological assays are ideally suited for this task, and the recent development of automated electrophysiology instrumentation affords medium throughput. Higher capacity assays amenable to studying sodium channel pharmacology include ligand binding, flux, and fluorescent assays.
Ion channels play a vital role in basic physiological functions such as generation of electrical activity in nerves and muscle, control of cardiac excitability, intracellular signaling, hormone secretion, cell proliferation, cell volume regulation, and many other biological processes. Because of their prevalence and the critical role they play in virtually all tissue types and organs, ion channels are also involved in a number of pathophysiological conditions. The recognized importance of ion channels in health and disease, combined with the potential to develop new drugs targeting ion channels in a broad range of diseases, has fueled the need to develop more suitable screening technologies accounting for their complex structure and function. Ion channels have been neglected as drug discovery targets because of the inability to study large number of compounds or validate large numbers of unknown or mutant ion channel genes using traditional ion channel screening technologies. Therefore, several efforts were undertaken to automate and improve the throughput of electrophysiological methods. In this chapter, we will review a number of the more standard ion channel screening technologies currently used, including: (1) radioligand binding assays, (2) fluorescent assays using membrane potential dyes, and (3) ion flux assays, and emphasize some of the advantages and shortcomings of these different approaches. We will then discuss automated patch clamp technologies that aim to automate and dramatically increase the throughput of the standard voltage clamp method, and offer a true archetype shift in ion channel drug discovery.
Pain sensation is encoded in the firing patterns of a variety of neurons throughout the body, and much of this signaling is dependent on the complement of expressed ion channel proteins. Ion channels, therefore, present excellent molecular targets for designing new therapeutic molecules to manage pain. In this review we discuss two classes of channels which are currently of interest to the pharmaceutical community in this area. The first are Kv7 potassium channels which encode the M-current. Opening of these channels tends to dampen neuronal excitability and pain signaling; therefore, agonists are of potential interest to treat pain. The second class is Cav3 calcium channels which encode T-type calcium currents. The opening of these channels typically promotes neuronal excitability and inhibitors could also be useful to treat various types of pain. The following review discusses the properties of theses two channel types along with recent advances in the identification of small molecule modulators.
The Kv1.5 channel subunit is the putative molecular correlate of the ultrarapidly activating delayed rectifier potassium current, I-Kur, detected in human atrial but not ventricular myocytes. Inhibition of Kv1.5 has been widely recognized as a potential therapeutic strategy for the treatment of atrial fibrillation (AF), a major clinical concern, especially in aging populations. This review centers on recent Kv1.5 drug discovery and development, in the context of action potential prolonging antiarrhythmics for the treatment of AF. Collectively, most novel agents are nonselective for Kv1.5. Preclinical along with very limited clinical efficacy data appear to validate Kv1.5 as an effective target to increase atrial refractoriness and to convert fibrillating atria to sinus rhythm. Functional characterization of Kv1.5-specific structural domains and novel assays can help discover better Kv1.5 drugs. Definite proof of concept for Kv1.5 inhibition as an effective antiarrhythmic strategy awaits agents with greater Kv1.5 selectivity and a more detailed characterization of I-Kur in human atria and the consequence of its inhibition.
The combination of green fluorescent protein mutants and fluorescence resonance energy transfer (FRET) forms a powerful tool for ion channel studies. A key to successful application of green fluorescent protein-based FRET is to reliably separate the FRET signal from various non-FRET fluorescence emissions that coexist in any experimental system. This chapter introduces a FRET quantification method that is based on fluorescence spectroscopic microscopy. Application of this "spectra FRET" method to both the confocal imaging of Xenopus oocytes and the epifluorescence imaging of culture cells is described. The fluorescence intensity ratio measurement, a complementary non-FRET method for identifying the channel subunit stoichiometry, is also discussed.
Potassium channels play an essential role in a wide range of biological processes, including cell volume regulation and the maintenance and control of electrical signals. With the advent of the structural era of ion channel biology, it has become critical to learn more about the functional properties of the prokaryotic channels, and this is the area in which genetic screens have become an increasingly useful approach. Here, we describe a bacteria-based complementation assay that we applied to investigate gating mutants of the prokaryotic K+ channel MthK, which was cloned from the archeon Methanobacterium thermoautotrophicum. The results demonstrated that heterologously expressed MthK is fully assembled and functional in Escherichia coli. This complementation assay should be useful in the initial identification of prokaryotic K+ channel mutants that result in altered channel function.
Voltage-gated potassium channels are ubiquitous and critical for life. They must fold and assemble correctly and target to appropriate sites in the plasma membrane. Failure to do so can lead to inappropriate targeting or function and to pathology. The methods described here were developed to assess in which compartment tertiary and quaternary structure acquisition occurs. The experimental strategies involve identifying quaternary and tertiary interfaces, engineering a pair of cysteines into a cysteine-free voltage-gated potassium channel protein, using bifunctional crosslinking agents, and using an assay of the crosslinked products to determine folding/assembly events. A biogenic intermediate (i.e., nascent chain attached to transfer RNA and the ribosome) is used to probe events inside and at the exit port of the ribosomal tunnel.
Extracellular recording of the action potential discharge of individual neurons has been an indispensable electrophysiological method for more than 50 yr. Although it requires relatively modest instrumentation, extracellular recording nevertheless provides critically important information concerning the patterning of intercellular communication in the nervous system. In 1996, Didier Pinault described “juxtacellular labeling” as “a novel and very effective single-cell labeling method” for revealing the morphology of extracellularly recorded neurons. Of particular interest for neuroscience is that juxtacellular labeling can be combined with immunocytochemistry and in situ hybridization histochemistry to reveal new and exciting information concerning the chemical phenotype of neurons whose electrophysiological properties have been characterized in vivo. By providing investigators with a means to “match” functional information from electrophysiological recordings with morphological and protein/gene expression data at the level of the single neuron, juxtacellular labeling has opened a new era in neuroscience research, one that holds the promise of an accelerated pace of discovery.
Exogenous expression of genes in mammalian neurons represents a substantial experimental challenge because of the low efficiency of commercially available liposomal transfection reagents for nondividing cells and considerable toxicity of viral transfection systems. In this chapter, we discuss application of the "biolistic" particle delivery system for heterologous expression of genes in primary neuron cultures. The method is based on the direct introduction of cDNA of interest into the nucleus by penetration with DNA-coated gold particles. With this approach, cDNA expression is independent of cell cycling and proliferation and is similar to intranuclear microinjection, with both avoiding cDNA delivery through the cytosol. Examples of successful transfection using PDS of rat superior cervical ganglion and trigeminal ganglion neurons are discussed.
Analysis of genetically engineered mice is crucial for our understanding of the in vivo function of genes and proteins in the whole organism. This includes inactivation of a gene or the generation of specific mutations. The development of knockout and transgenic technologies in the mouse, therefore, represents a powerful tool for elucidating gene function, for modeling of human diseases, and potentially for the evaluation of drugs. In particular, conditional gene targeting applying the Cre/loxP-mediated recombination system is increasingly used to evaluate the role of the gene of interest in a cell-type-specific or even inducible manner. The experimental steps start with the characterization of the gene locus, followed by construction of a vector, gene targeting in ES cells, and establishment of mouse lines carrying the desired mutation. These are then bred to transgenic mice expressing Cre recombinase in a tissue-specific manner, thus allowing gene inactivation in a cell type of interest.
Probing ion channel structure-function and regulation in native tissue can, in some instances, be experimentally challenging or impractical. To facilitate discovery and increase experimental flexibility, our laboratory routinely reconstitutes recombinant ion channels in a mammalian expression system quantifying channel activity with patch clamp electrophysiology. Here, we describe investigation of the human epithelial Na+ channel heterologously expressed in Chinese hamster ovary cells.
The amiloride-sensitive epithelial Na+ channel (ENaC) is typically composed of three structurally related subunits termed alpha, beta, and gamma. We describe methods to determine the functional subunit stoichiometry of ENaC based on a biophysical approach that was first introduced in 1991 to determine the subunit stoichiometry of a voltage-gated K+ channel. The strategy is to analyze channel sensitivity to a specific blocker when various mixtures of block-sensitive and blocker-insensitive subunits are coexpressed in a heterologous expression system. Details related to the expression of wild type and mutant ENaCs in Xenopus oocytes and the examination of blocker sensitivity by two-electrode voltage clamp, as well as analysis of data are provided.
Ion channels are integral membrane proteins that control transmembrane ion fluxes to regulate membrane potential, cell excitability, and ion transport. Membrane phospho-lipids containing phosphoinositides have recently emerged as important regulators of many ion channels, including inward rectifier K + channel, voltage-gated K + and Ca 2+ channels, transient receptor potential channels, and intracellular inositol-1,4,5-trisphos-phate receptor ion channels. Discussed here are several methods for studying regulation of ion channels by phospholipids.
Overexpression of proteins is a powerful way to determine their function. Until recently, the low efficiency of neuronal transfection has made it difficult to use overexpression and structure-function studies to investigate the role of neuronal proteins in their native environment. The development of neurotrophic viral systems has overcome the obstacle of low efficiency and allows for unprecedented opportunities to use biochemical and electrophysiological techniques to assess the effects of overexpressing wild-type or mutant proteins in neurons. Here, a general protocol for the production of replicationdeficient Semliki Forest virus constructs directing the overexpression of proteins of interest in cultured mammalian neurons is described.
Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. However, this method only measures the sum of the activity of all plasma membrane-bound channels. Therefore, this measurement cannot discriminate between effects on channel density and individual channel activity. To address this shortcoming, we have developed a simple assay to detect changes of membrane-bound channel density in intact oocytes. This nonradioactive assay relies on specific antibody binding in whole live cells utilizing a simple spectrophotometric measurement. This assay is linear over a wide range of channel expression levels and provides a simple cost-effective way of monitoring changes of membrane-bound channel density. Moreover, when the heterologous proteins poorly express at the plasma membrane, this method becomes advantageous to complex biochemical cell fractionation.
