Molecular targets for autoimmune and genetic disorders of neuromuscular transmission.

The neuromuscular junction is the target of a variety of autoimmune, neurotoxic and genetic disorders, most of which result in muscle weakness. Most of the diseases, and many neurotoxins, target the ion channels that are essential for neuromuscular transmission. Myasthenia gravis is an acquired autoimmune disease caused in the majority of patients by antibodies to the acetylcholine receptor, a ligand-gated ion channel. The antibodies lead to loss of acetylcholine receptor, reduced efficiency of neuromuscular transmission and muscle weakness and fatigue. Placental transfer of these antibodies in women with myasthenia can cause fetal or neonatal weakness and occasionally severe deformities. Lambert Eaton myasthenic syndrome and acquired neuromyotonia are caused by antibodies to voltage-gated calcium or potassium channels, respectively. In the rare acquired neuromyotonia, reduced repolarization of the nerve terminal leads to spontaneous and repetitive muscle activity. In each of these disorders, the antibodies are detected by immunoprecipitation of the relevant ion channel labelled with radioactive neurotoxins. Genetic disorders of neuromuscular transmission are due mainly to mutations in the genes for the acetylcholine receptor. These conditions show recessive or dominant inheritance and result in either loss of receptors or altered kinetics of acetylcholine receptor channel properties. Study of these conditions has greatly increased our understanding of synaptic function and of disease aetiology.     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

New insights into TRP channels: Interaction with pattern recognition receptors

An increasing number of studies have implicated that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms in response to pathogens or tissue injury, which is performed via germ-line encoded pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) or dangers-associated molecular patterns (DAMPs). Intracellular pathways linking immune and inflammatory response to ion channel expression and function have been recently identified. Among ion channels, transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes. In this review, we summarize current knowledge about classifications, functions, and interactions of TRP channels and PRRs, which may provide new insights into their roles in the pathogenesis of inflammatory diseases.     

Genetic strategies to analyze primary TRP channel-expressing cells in mice

Transient receptor potential (TRP) ion channels regulate fundamental biological processes throughout the body. TRP channel dysfunction has been causally linked to a number of disease states and thus establishes these channels as promising therapeutic targets. In order to dissect the physiological role of individual TRP channels in specific tissues, a detailed understanding of the expression pattern of the different TRP channels throughout the organism is essential. We provide an overview of recent efforts to generate novel TRP channel reporter mouse strains for all 28 TRP channels encoded in the mouse genome to understand expression of these channels with a single-cell resolution in an organism-wide manner. The reporter mice will enable both the visualization and manipulation of all primary TRP channel-expressing cells allowing an unprecedented wealth in variety to investigate TRP channel function in vivo. As proof of principle, we provide preliminary results documenting TRPM5 expression throughout the entire body of juvenile and adult mice.     

Physiological and Pathological Functions of Mechanosensitive Ion Channels

Rapid sensation of mechanical stimuli is often mediated by mechanosensitve ion channels. Their opening results from conformational changes induced by mechanical forces. It leads to membrane permeation of selected ions and thereby to electrical signaling. Newly identified mechanosensitive ion channels are emerging at an astonishing rate, including some that are traditionally assigned for completely different functions. In this review, we first provide a brief overview of ion channels that are known to play a role in mechanosensation. Next, we focus on three representative ones, including the transient receptor potential channel V4 (TRPV4), Kv1.1 voltage-gated potassium (Kv) channel, and Piezo channels. Their structures, biophysical properties, expression and targeting patterns, and physiological functions are highlighted. The potential role of their mechanosensation in related diseases is further discussed. In sum, mechanosensation appears to be achieved in a variety of ways by different proteins and plays a fundamental role in the function of various organs under normal and abnormal conditions.     

Channelopathies of inwardly rectifying potassium channels.

Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine.     

Modulation and genetic identification of the M channel

Potassium channels constitute a superfamily of the most diversified ion channels, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, transmitter release, cell proliferation and cell degeneration. The M-type channel is a unique ligand-regulated and voltage-gated K(+) channel showing distinct physiological and pharmacological characteristics. This review will cover some important progress in the study of M channel modulation, particularly focusing on membrane transduction mechanisms. The K(+) channel genes corresponding to the M channel have been identified and will be reviewed in detail.It has been a long journey since the discovery of M current in 1980 to our present understanding of the mysterious mechanisms for M channel modulation; a journey which exemplifies tremendous achievements in ion channel research and exciting discoveries of elaborate modulatory systems linked to these channels. While substantial evidence has accumulated, challenging questions remain to be answered.     

The transient receptor potential family of ion channels

The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca²⁺ selective, some are even permeable for highly hydrated Mg²⁺ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca²⁺ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca²⁺ and Mg²⁺), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca²⁺ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases.     

Oxidative stress-modulated TRPM ion channels in cell dysfunction and pathological conditions in humans

The transient receptor potential melastatin (TRPM) protein family is an extensive group of ion channels expressed in several types of mammalian cells. Many studies have shown that these channels are crucial for performing several physiological functions. Additionally, a large body of evidence indicates that these channels are also involved in numerous human diseases, known as channelopathies.A characteristic event frequently observed during pathological states is the raising in intracellular oxidative agents over reducing molecules, shifting the redox balance and inducing oxidative stress. In particular, three members of the TRPM subfamily, TRPM2, TRPM4 and TRPM7, share the remarkable feature that their activities are modulated by oxidative stress.Because of the increase in oxidative stress, these TRPM channels function aberrantly, promoting the onset and development of diseases.Increases, absences, or modifications in the function of these redox-modulated TRPM channels are associated with cell dysfunction and human pathologies. Therefore, the effect of oxidative stress on ion channels becomes an essential part of the pathogenic mechanism. Thus, oxidative stress-modulated ion channels are more susceptible to generating pathological states than oxidant-independent channels.This review examines the most relevant findings regarding the participation of the oxidative stress-modulated TRPM ion channels, TRPM2, TRPM4, and TRPM7, in human diseases. In addition, the potential roles of these channels as therapeutic tools and targets for drug design are discussed.     

Trends in ion channel drug discovery: advances in screening technologies

Ion channels mediate and regulate crucial electrical functions throughout the body. They are therapeutic drug targets for a variety of disorders and, in some cases, the direct cause of unwanted side-effects. Advances in medical genetics have increased our knowledge of ion channel structure–function relationships and identified disease-causing mutations in ion channel genes. The recognized importance of these proteins in health and disease has led to an active search for ion channel targets in the multi-billion-dollar worldwide drug discovery market. Trends in ion channel screening technologies have focused on increasing throughput and enhancing information content of assays through electrophysiological approaches. The ability to study ion channels by voltage clamp and their time-, voltage- and state-dependent drug interactions with enhanced throughput will ultimately play a key role in the development of novel, safe ion channel-targeted drugs.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca (2+) is essential for cardiomyocyte function, and alterations in proteins that regulate Ca (2+) influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca (2+) channels are one pathway of Ca (2+) entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Cav3.1 (α1G) and Cav3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca (2+) channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Cav3.1 and Cav3.2 Ca (2+) channel expression in the pathological setting. The putative association of T-type Ca (2+) channels with Ca (2+) dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.     

Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.     

Hypotonicity induces TRPV4-mediated nociception in rat

We hypothesized that TRPV4, a member of the transient receptor family of ion channels, functions as a sensory transducer for osmotic stimulus-induced nociception. We found that, as expected for a transducer molecule, TRPV4 protein is transported in sensory nerve distally toward the peripheral nerve endings. In vivo single-fiber recordings in rat showed that hypotonic solution activated 54% of C-fibers, an effect enhanced by the hyperalgesic inflammatory mediator prostaglandin E2. This osmotransduction causes nociception, since administration of a small osmotic stimulus into skin sensitized by PGE2 produced pain-related behavior. Antisense-induced decrease in expression of TRPV4 confirmed that the channel is required for hypotonic stimulus-induced nociception. Thus, we conclude that TRPV4 can function as an osmo-transducer in primary afferent nociceptive nerve fibers. Because this action is enhanced by an inflammatory mediator, TRPV4 may be important in pathological states and may be an attractive pharmacological target for the development of novel analgesics.     

Calcium release-activated calcium channels and pain

Calcium release-activated calcium (CRAC) channels are unique among ion channels that are activated in response to depletion of intracellular calcium stores and are highly permeable to Ca²⁺ compared to other cations. CRAC channels mediate an important calcium signal for a wide variety of cell types and are well studied in the immune system. They have been implicated in a number of disorders such as immunodeficiency, musculosketal disorders and cancer. There is growing evidence showing that CRAC channels are expressed in the nervous system and are involved in pathological conditions including pain. This review summarizes the expression, distribution, and function of the CRAC channel family in the dorsal root ganglion, spinal cord and some brain regions, and discusses their functional significance in neurons and glial cells and involvement in nociception and chronic pain. Although further studies are needed to understand how these channels are activated under physiological conditions, the recent findings indicate that the CRAC channel Orai1 is an important player in pain modulation and could represent a new target for pathological pain.     

Investigations of the Contribution of a Putative Glycine Hinge to Ryanodine Receptor Channel Gating

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K⁺-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K⁺ channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G⁴⁸⁶⁴LIIDA⁴⁸⁶⁹ in RyR2) analogous to the glycine hinge motif present in many K⁺ channels. Gating in these K⁺ channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K⁺ channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K⁺ channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.     

Inflammation-induced “Channelopathies” in the gastrointestinal smooth muscle

Inflammation markedly alters the motility patterns of the gastrointestinal tract, resulting mostly in decreased excitability of smooth muscle. There is emerging evidence indicating that inflammation alters ion channel expression and function of smooth muscle cells. In this review we summarize studies defining the mechanisms affecting contractile and electrical activity of gastrointestinal smooth muscle. We have focused on the evidence for decreased calcium channel conductance and alterations in the intracellular signaling mechanisms and discuss the role of muscarinic receptor activation in models of gastrointestinal inflammation. We propose that some of the clinical symptoms of altered smooth muscle contraction in pathogenesis of gut disorders such as inflammatory bowel disease may be regulated at the level of the ion channel.     

Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors).

Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.     

A structural overview of the ion channels of the TRPM family

The TRPM (transient receptor potential melastatin) family belongs to the superfamily of TRP cation channels. The TRPM subfamily is composed of eight members that are involved in diverse biological functions such as temperature sensing, inflammation, insulin secretion, and redox sensing. Since the first cloning of TRPM1 in 1998, tremendous progress has been made uncovering the function, structure, and pharmacology of this family. Complete structures of TRPM2, TRPM4, and TRPM8, as well as a partial structure of TRPM7, have been determined by cryo-EM, providing insights into their channel assembly, ion permeation, gating mechanisms, and structural pharmacology. Here we summarize the current knowledge about channel structure, emphasizing general features and principles of the structure of TRPM channels discovered since 2017. We also discuss some of the key unresolved issues in the field, including the molecular mechanisms underlying voltage and temperature dependence, as well as the functions of the TRPM channels’ C-terminal domains.     

Polymodal Regulation of NMDA Receptor-Channels

Glutamate-activated N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels which mediate synaptic transmission, long-term potentiation, synaptic plasticity and neurodegeneration via conditional Ca2+ signalling. Recent crystallographic studies have focussed on solving the structural determinant of the ligand binding within the core region of NR1 and NR2 subunits. Future structural analysis will help to understand the mechanism of native channel activation and regulation during synaptic transmission. A number of NMDA receptor ligands have been identified which act as positive or negative modulators of receptor function. There is evidence that the lipid bilayer can further regulate the activity of the NMDA receptor channels. Modulators of NMDA receptor function offer the potential for the development of novel therapeutics to target neurological disorders associated with this family of glutamate ion channel receptors. Here, we review the recent literature concerning structural and functional properties, as well as the physiological and pathological roles of NMDA receptor channels.