Domenico Tricarico

Ion channel pharmacology

SummaryBecause ion channels are involved in many cellular processes, drugs acting on ion channels have long been used for the treatment of many diseases, especially those affecting electrically excitable tissues. The present review discusses the pharmacology of voltage-gated and neurotransmitter-gated ion channels involved in neurologic diseases, with emphasis on neurologic channelopathies. With the discovery of ion channelopathies, the therapeutic value of many basic drugs targeting ion channels has been confirmed. The understanding of the genotype—phenotype relationship has highlighted possible action mechanisms of other empirically used drugs. Moreover, other ion channels have been pinpointed as potential new drug targets. With regards to therapy of channelopathies, experimental investigations of the intimate drug—channel interactions have demonstrated that channel mutations can either increase or decrease affinity for the drug, modifying its potential therapeutic effect. Together with the discovery of channel gene polymorphisms that may affect drug pharmacodynamics, these findings highlight the need for pharmacogenetic research to allow identification of drugs with more specific effects on channel isoforms or mutants, to increase efficacy and reduce side effects. With a greater understanding of channel genetics, structure, and function, together with the identification of novel primary and secondary channelopathies, the number of ion channel drugs for neurologic channelopathies will increase substantially.

Impairment of skeletal muscle adenosine triphosphate–sensitive K+ channels in patients with hypokalemic periodic paralysis

The adenosine triphosphate (ATP)-sensitive K+ (KATP) channel is the most abundant K+ channel active in the skeletal muscle fibers of humans and animals. In the present work, we demonstrate the involvement of the muscular KATP channel in a skeletal muscle disorder known as hypokalemic periodic paralysis (HOPP), which is caused by mutations of the dihydropyridine receptor of the Ca2+ channel. Muscle biopsies excised from three patients with HOPP carrying the R528H mutation of the dihydropyridine receptor showed a reduced sarcolemma KATP current that was not stimulated by magnesium adenosine diphosphate (MgADP; 50-100 microM) and was partially restored by cromakalim. In contrast, large KATP currents stimulated by MgADP were recorded in the healthy subjects. At channel level, an abnormal KATP channel showing several subconductance states was detected in the patients with HOPP. None of these were surveyed in the healthy subjects. Transitions of the KATP channel between subconductance states were also observed after in vitro incubation of the rat muscle with low-K+ solution. The lack of the sarcolemma KATP current observed in these patients explains the symptoms of the disease, i.e., hypokalemia, depolarization of the fibers, and possibly the paralysis following insulin administration.

Acetazolamide opens the muscular K Ca 2+ channel: A novel mechanism of action that may explain the therapeutic effect of the drug in hypokalemic periodic paralysis

Acetazolamide is a thiazide derivative clinically used in skeletal muscle disorders related to altered K+ homeostasis such as the periodic paralyses. The mechanism of action responsible for the therapeutic effects of the drug is still unknown, however. In the present work, we investigated the mechanism of action of acetazolamide in the K–deficient diet rat, an animal model of human hypokalemic periodic paralysis (hypoPP). The in vivo administration of 2.8‐ and 5.6‐mg/kg−1/day−1 concentrations of acetazolamide to K–deficient diet rats prevented paralysis and depolarization of the fibers induced by insulin. In the acetazolamide‐treated animals, intense sarcolemma Ca2+‐activated K+ channel (K  Ca 2+ ) activity was recorded. Acetazolamide also restored the serum K+ levels to control values. The concentrations of acetazolamide needed to enhance the K  Ca 2+ current by 50% in vitro were 6.17 and 4.01 × 10−6 M at −60 and +30 mV of membrane potentials, respectively. In normokalemic animals, the thiazide derivative enhanced the K  Ca 2+ current with similar efficacy. Our data demonstrate that the therapeutic effects of acetazolamide in the K–deficient diet rats and possibly in human hypokalemic periodic paralysis patients can be mediated by activation of the K  Ca 2+ channel. Ann Neurol 2000;48:304–312

Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats.

Carbonic‐anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch‐clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+‐activated‐K+ (BK) channel of K+‐deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10− 6M)>BFT(DE50=5.93x10− 5M)>ETX(DE50=1.17x10− 4M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra‐molecular hydrogen bond with calculated inter‐atomic distances ranging between 1.82 A° and 3.01 A° and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.

Enantiomers of clofibric acid analogs have opposite actions on rat skeletal muscle chloride channels

The S-(−) isomers of a series of clofibric acid analogs produced only a block of chloride conductance of rat skeletal muscle fibers with increasing concentrations until block was nearly complete. The R-(+) isomers, on the other hand, at low concentrations increased chloride conductance by as much as 9% to 39% and at higher concentrations decreased chloride conductance, but never by more than 27% of the control value. The actions of the enantiomeric pairs to either produce or inhibit myotonic excitability paralleled their ability to block or increase chloride conductance, respectively.

Taurine and skeletal muscle disorders.

Taurine is abundantly present in skeletal muscle. We give evidence that this amino acid exerts both short-term and long-term actions in the control of ion channel function and calcium homeostasis in striated fibers. Short-term actions can be estimated as the ability of this amino acid to acutely modulate both ion channel gating and the function of the structures involved in calcium handling. Long-term effects can be disclosed in situations of tissue taurine depletion and are likely related to the ability of the intracellular taurine to control transducing pathways as well as homeostatic and osmotic equilibrium in the tissue. The two activities are strictly linked because the intracellular level of taurine modulates the sensitivity of skeletal muscle to the exogenous application of taurine. Myopathies in which ion channels are directly or indirectly involved, as well as inherited or acquired pathologies characterized by metabolic alterations and change in calcium homeostasis, are often correlated with change in muscle taurine concentration and consequently with an enhanced therapeutic activity of this amino acid. We discuss both in vivo and in vitro evidence that taurine, through its ability to control sarcolemmal excitability and muscle contractility, can prove beneficial effects in many muscle dysfunctions.

Therapeutic Approaches to Ion Channel Diseases

More than 400 genes are known that encode ion channel subunits. In addition, alternative splicing and heteromeric assembly of different subunits increase tremendously the variety of ion channels. Such many channels are needed to accomplish very complex cellular functions, whereas dysfunction of ion channels are key events in many pathological processes. The recent discovery of ion channelopathies, which, in its more stringent definition, encloses monogenic disorders due to mutations in ion channel genes, has largely contributed to our understanding of the function of the various channel subtypes and of the role of ion channels in multigenic or acquired diseases. Last but not least, ion channels are the main targets of many drugs already used in the clinics. Most of these drugs were introduced in therapy based on the experience acquired quite empirically, and many were discovered afterward to target ion channels. Now, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction. In this review, we first focus on the pharmacotherapy of ion channel diseases, which includes many drugs targeting ion channels. Then, we describe the molecular pharmacology of ion channels, including the more recent advancement in drug development. Among the newest aspect of ion channel pharmacology, we draw attention to how polymorphisms or mutations in ion channel genes may modify sensitivity to drugs, opening the way toward the development of pharmacogenetics.

Aging and chloride channel regulation in rat fast-twitch muscle fibres

By the use of pharmacological tools, we tested the hypothesis that age-related alterations in the regulatory pathways of chloride channels might contribute to the lowered chloride conductance (GCl) found in skeletal muscle of aged rats. The restingGCl of extensor digitorum longus (EDL) muscles from adult rats either young (3–4 months old) or aged (29 months old) was measured by means of computerized intracellular microelectrode recordings. In EDL muscle from 3 to 4-month-old rats, 4-β-phorbol 12,13-dibutyrate (4-β-PDB), a direct activator of protein kinase C (PKC), decreasedGCl in a concentration-dependent manner. The same effect was exerted by cholera toxin. The effects of both the phorbol ester and cholera toxin were inhibited by staurosporine, thus indicating that either direct or indirect (via G protein) activation of PKC accounts for the decrease ofGCl. An increase of cytosolic Ca2+ by the ionophore A23187 also significantly decreasedGCl by 25%. In EDL muscles from aged rats, 4-β-PDB was 20-fold more potent in blockingGCl than in muscles from younger controls, and the ionophore blockedGCl by 40%. On the other hand, cholera toxin was ineffective. Our findings support the hypothesis that in fast-twitch muscle the regulation of chloride channels by PKC and Ca2+ is a target of the aging process.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.

Major Channels Involved In Neuropsychiatric Disorders And Therapeutic Perspectives

Voltage-gated ion channels are important mediators of physiological functions in the central nervous system. The cyclic activation of these channels influences neurotransmitter release, neuron excitability, gene transcription, and plasticity, providing distinct brain areas with unique physiological and pharmacological response. A growing body of data has implicated ion channels in the susceptibility or pathogenesis of psychiatric diseases. Indeed, population studies support the association of polymorphisms in calcium and potassium channels with the genetic risk for bipolar disorders (BPDs) or schizophrenia. Moreover, point mutations in calcium, sodium, and potassium channel genes have been identified in some childhood developmental disorders. Finally, antibodies against potassium channel complexes occur in a series of autoimmune psychiatric diseases. Here we report recent studies assessing the role of calcium, sodium, and potassium channels in BPD, schizophrenia, and autism spectrum disorders, and briefly summarize promising pharmacological strategies targeted on ion channels for the therapy of mental illness and related genetic tests.