Paola Imbrici

Differential pH sensitivity of Kir4.1 and Kir4.2 potassium channels and their modulation by heteropolymerisation with Kir5.1

1 The inwardly rectifying potassium channel Kir5.1 appears to form functional channels only by coexpression with either Kir4.1 or Kir4.2. Kir4.1‐Kir5.1 heteromeric channels have been shown to exist in vivo in renal tubular epithelia. However, Kir5.1 is expressed in many other tissues where Kir4.1 is not found. Using Kir5.1‐specific antibodies we have localised Kir5.1 expression in the pancreas, a tissue where Kir4.2 is also highly expressed. 2 Heteromeric Kir5.1‐Kir4.1 channels are significantly more sensitive to intracellular acidification than Kir4.1 currents. We demonstrate that this increased sensitivity is primarily due to modulation of the intrinsic Kir4.1 pH sensitivity by Kir5.1. 3 Kir4.2 was found to be significantly more pH sensitive (pKa= 7.1) than Kir4.1 (pKa= 5.99) due to an additional pH‐sensing mechanism involving the C‐terminus. As a result, coexpression with Kir5.1 does not cause a major shift in the pH sensitivity of the heteromeric Kir4.2‐Kir5.1 channel. 4 Cell‐attached single channel analysis of Kir4.2 revealed a channel with a high open probability (Po > 0.9) and single channel conductance of ˜25 pS, whilst coexpression with Kir5.1 produced novel bursting channels (Po < 0.3) and a principal conductance of ˜54 pS with several subconductance states. 5 These results indicate that Kir5.1 may form heteromeric channels with Kir4.2 in tissues where Kir4.1 is not expressed (e.g. pancreas) and that these novel channels are likely to be regulated by changes in intracellular pH. In addition, the extreme pH sensitivity of Kir4.2 has implications for the role of this subunit as a homotetrameric channel.

Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4‐1.1/Kvβ1.1 and Kv1.4‐1.1/Kvβ1.2

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage‐gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed‐rectifier function of the channel. Shaker‐like channels of different cell types may be formed by unique hetero‐oligomeric complexes comprising Kv1.1, Kv1.4 and Kvβ1.x subunits. Here we show that the human Kvβ1.1 and Kvβ1.2 subunits modulated the functional properties of tandemly linked Kv1.4‐1.1 wild‐type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N‐type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4‐1.1, Kv1.4‐1.1/Kvβ1.1 and Kv1.4‐1.1/Kvβ1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion‐conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N‐type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady‐state inactivation to more positive potentials. The results demonstrate that the human Kvβ1.1 and Kvβ1.2 subunits regulate the proportion of wild‐type Kv1.4‐1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements.

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.

Modification by ageing of the tetrodotoxin-sensitive sodium channels in rat skeletal muscle fibres

Ageing leads to an impairment of muscle performance that may result from alteration of sarcolemma excitability. Therefore, we compare sodium channels of native fast-twitch skeletal muscle fibres of 21-26-month-old aged rats and 4-6-month-old young-adult rats, using the patch-clamp method. Extrajunctional sarcolemma of aged-rat fibres presented a higher sodium current density than that of young-rat fibres, which resulted from the presence of a higher number of available channels per membrane area. Open probability and availability voltage-dependence of sodium channels were similar in aged- and young-rat fibres, but permeation property was altered during ageing: aged-rat muscles showed a bimodal distribution of fibres with two values of sodium-channel conductance measured between -40 and 0 mV; a young phenotype with a conductance close to 18 pS overlapping that found in young-rat fibres and an aged phenotype with a lower approximately half conductance. Current-voltage curves extended to -60 and +20 mV showed that the aged-phenotype conductance level resulted from an outward rectification occurring in these aged-rat fibres. Furthermore, in these aged-rat fibres belonging to the aged phenotype, ensemble average sodium currents showed slower activation and inactivation kinetics. Sodium currents of the two phenotypes were blocked by 100 nM tetrodotoxin, therefore excluding possible denervation effect. These age-related modifications in sodium current may contribute to the alteration of muscle excitability and function observed during the ageing process.

Genetic inactivation of KCNJ16 identifies Kir5.1 as an important determinant of neuronal PCO2/pH sensitivity

The molecular identity of ion channels which confer PCO2/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO2 chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16(+/+) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16(−/−) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO2/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.

A NOVEL KCNA1 MUTATION IDENTIFIED IN AN ITALIAN FAMILY AFFECTED BY EPISODIC ATAXIA TYPE 1

Episodic ataxia type 1 (EA1) is a rare human neurological syndrome characterized by continuous myokymia and attacks of generalized ataxia that can be triggered by abrupt movements, emotional stress and fatigue. An Italian family has been identified where related members displayed continuous myokymia, episodes of ataxia, attacks characterized by myokymia only, and neuromyotonia. A novel missense mutation (F414C), in the C-terminal region of the K(+) channel Kv1.1, was identified in the affected individuals. The mutant homotetrameric channels were non-functional in Xenopus laevis oocytes. In addition, heteromeric channels resulting from the co-expression of wild-type Kv1.1 and Kv1.1(F414C), or wild-type Kv1.2 and Kv1.1(F414C) subunits displayed reduced current amplitudes and altered gating properties. This indicates that the pathogenic effect of this KCNA1 mutation is likely to be related to the defective functional properties we have identified.

Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

ClC-1 chloride channels: state-of-the-art research and future challenges

The voltage-dependent ClC-1 chloride channel belongs to the CLC channel/transporter family. It is a homodimer comprising two individual pores which can operate independently or simultaneously according to two gating modes, the fast and the slow gate of the channel. ClC-1 is preferentially expressed in the skeletal muscle fibers where the presence of an efficient Cl- homeostasis is crucial for the correct membrane repolarization and propagation of action potential. As a consequence, mutations in the CLCN1 gene cause dominant and recessive forms of myotonia congenita (MC), a rare skeletal muscle channelopathy caused by abnormal membrane excitation, and clinically characterized by muscle stiffness and various degrees of transitory weakness. Elucidation of the mechanistic link between the genetic defects and the disease pathogenesis is still incomplete and, at this time, there is no specific treatment for MC. Still controversial is the subcellular localization pattern of ClC-1 channels in skeletal muscle as well as its modulation by some intracellular factors. The expression of ClC-1 in other tissues such as in brain and heart and the possible assembly of ClC-1/ClC-2 heterodimers further expand the physiological properties of ClC-1 and its involvement in diseases. A recent de novo CLCN1 truncation mutation in a patient with generalized epilepsy indeed postulates an unexpected role of this channel in the control of neuronal network excitability. This review summarizes the most relevant and state-of-the-art research on ClC-1 chloride channels physiology and associated diseases.