Abstracts

Studies of multimodal gating of the sodium channel

Richard D. Keynes

Chandler and Meves found that in squid axons perfused with NaF a small flow of Na+ ions persisted in the inactivated state, and that the Na+ channel therefore has more than one open state. Studies by Correa and Bezanilla on single patches in squid axons showed that such steady currents arose from reopening of the channel at a relatively low frequency. Currents with comparable properties are generated in mammalian brain cells and elsewhere. The existence of a third mode of gating was established by Patlak and Ortiz when they showed that in frog muscle fibres there were occasionally quite large bursts of late openings. Again, similar behaviour has been observed in other types of muscle and also in brain cells. It is suggested that the voltage gating of all ionic channels basically involves a screw-helical mechanism, operating in steps each transferring unit charge. For segment S4 in domain IV of Na+ channels, three charges have to be transferred to reach the initial open state, and a fourth for fast inactivation to take place. The single late openings in the inactivated steady state may be explained by the transfer of a fifth charge in IVS4, while the larger bursts of reopening involve a modulation of the mechanism of fast inactivation.

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©2001 The Novartis Foundation

Molecular bases for function in sodium channels

Richard Horn

Department of Physiology, Institute of Hyperexcitability, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107, USA

Na+ channels earned their unique role in excitable cells because of two functional properties, finely honed by evolution. The first is their exquisite sensitivity to small changes of membrane potential: a depolarization of only 10 mV can increase open probability by as much as two orders of magnitude. The second is the rapidity with which they respond to changes of membrane potential: their gates begin to open tens of microseconds after a depolarization. These features are built into two sets of moving parts: voltage sensors that respond directly to changes of membrane potential, and gates that open and close in response to voltage sensor movement. We have explored these movements using a combination of electrophysiology, site-directed mutagenesis, cysteine accessibility scanning and photoactivated cross-linking using a bifunctional cysteine reagent. The main voltage sensors of Na+ channels are four homologous S4 segments, each of which has a unique functional role. These transmembrane segments are almost completely surrounded by hydrophilic crevices. The membrane electric field moves these positively charged helices through a short, hydrophobic 'gating pore'. The minimum contact between an S4 segment and its gating pore insure that a small movement can rapidly move several of its charged residues across the electric field.

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©2001 The Novartis Foundation

Diverse functions and dynamic expression of neuronal sodium channels

Stephen G. Waxman, Theodore R. Cummins, Joel A. Black and Sulayman Dib-Hajj

Nearly a dozen genes encode different Na+ channels, sharing a common overall motif but with subtly different amino acid sequences. Physiological signatures have now been established for some Na+ channels and it is clear that, from a functional point of view, Na+ channels are not all the same: different channels can have different physiological characteristics, and they can play different roles in the physiology of excitable cells. Moreover, the expression of Na+ channels within neurons is not a static process. Plasticity of Na+ channel gene expression occurs in the normal nervous system, where it accompanies transitions between different physiological states (e.g. low-frequency versus high-frequency firing states) in some types of neurons. Maladaptive changes in Na+ channel gene expression also occur in some pathological neurons. For example, transection of the peripheral axons of spinal sensory neurons triggers down-regulation of some Na+ channel genes and up-regulation of others, resulting in changes in Na+ current expression that produce hyperexcitability, thereby contributing to chronic pain. There is also recent evidence for the expression of normally silent Na+ channel genes in Purkinje cells in experimental models of demyelinating diseases and in a human disease, multiple sclerosis; this dysregulation of Na+ channel expression may interfere with neuronal function in these disorders. The diversity and dynamic nature of Na+ channel expression introduce a high degree of complexity into the nervous system and present challenges for neuroscientists. In addition, they may present therapeutic opportunities as selective modulators for various Na+ channel subtypes become available.

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©2001 The Novartis Foundation

Enhanced transmission of glutamate current flowing from the dendrite to the soma in rat neocortical layer 5 neurons

Wayne E. Crill, Peter C. Schwindt and John C. Oakley

The presence of tetrodotoxin (TTX)-sensitive slowly-inactivating Na+ channels in the dendrites of neocortical layer 5 neurons was tested by focal iontophoresis of glutamate on the dendrite while voltage clamping the soma and proximal dendrite. The glutamate-transmitted current was measured with the voltage clamp circuit. When the soma was depolarized the transmitted current increased indicating voltage-dependent properties in the dendrite. Over 50% of this increased voltage-dependent was blocked by TTX indicating a large portion of the enhanced dendro-somatic current was caused non-inactivating Na+ channel inward rectification. The glutamate-transmitted current measured with voltage clamp of the soma at firing level was equal to the effective glutamate measured during repetitive firing..

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©2001 The Novartis Foundation

Mutations of voltage-gated sodium channels in movement disorders and epilepsy

Miriam H. Meisler, Jennifer A. Kearney, Leslie K. Sprunger, Bryan T. MacDonald, David A. Buchner and Andrew Escayg

Spontaneous and induced mutations of neuronal Na+ channels in human patients and mutant mice result in a broad range of neurological disease. Epilepsy, a disorder of neuronal hyperexcitability, has been associated with delayed inactivation of SCN2A in mice, and with altered kinetics of SCN1A in human patients. Movement disorders including tremor, ataxia, dystonia and paralysis result have been observed in mice with mutations of SCN8A. Electrophysiological recordings from neurons isolated from mice with mutations in individual channels reveal the contributions of each channel to in vivo firing patterns. In addition to monogenic disease, Na+ channel mutations are likely to contribute to polygenic disease susceptibility and to normal variation in neuronal function. Advances in molecular methods coupled with genomic sequences from the Human Genome Project will permit identification of many new patient mutations and generation of animal models to dissect their physiological and cellular consequences.

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©2001 The Novartis Foundation

Channelopathies: episodic disorders of the nervous system

Louis Ptacek

The field of channelopathies is a newly recognized group of disorders named for the site of the molecular defects-voltage- and ligand-gated ion channels. While voltage-gated ion channel mutants have been recognized for some time in organisms such as Drosophila, the first channelopathy in humans was first reported within the last decade. The recognition of this group of disorders began with definition of the molecular basis of a group of unusual muscle disorders called the periodic paralysis and non-dystrophic myotonias. Interestingly, this group of muscle disorders share some interesting phenotypic features with a number of seemingly disparate human diseases that involve not only skeletal muscle, but also brain and heart. Some similarities that exist among these different disorders include their episodic nature, similarities with regard to factors that precipitate attacks, therapeutic agents which can help to treat or prevent attacks, and in some cases, a degenerative component that arises in addition to the episodic attacks. The study of these diseases, along with the recognition of common clinical and pathophysiological themes among these disorders has led to tremendous growth in our understanding of these diseases and the hope of developing better therapies.

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©2001 The Novartis Foundation

Sodium channel gene expression and epilepsy

Jeffrey L. Noebels

Na+ channelopathies that prolong membrane depolarization lead to neuronal bursting, abnormal network synchronization and various patterns of episodic neurological disorders, including epilepsy. Two distinct pathways exist for generating epileptic phenotypes based on inherited disorders of voltage-gated Na+ ion channels. The first pathway is direct, involving mutations in genes encoding the pore-forming a1 and regulatory b subunits of the channel that directly alter current amplitude or kinetics. These mutations favour repetitive firing and network hyperexcitability, although often the circuits most vulnerable to functional alterations are not easy to identify and the emergent clinical phenotypes are difficult to predict. The second pathway involves mutation of other genes that lead to downstream modifications in Na+ channel expression. Two clinically relevant examples of localization-related vulnerability in brain are described that illustrate how specific phenotypes arise from both direct and secondary pathways. Selective expression of the cardiac SCN5A channel within limbic regions of brain may explain why mutation of the gene for this tetrodotoxin-insensitive current may be associated with seizures. Ectopic expression of type II Na+ channels along axonal internodes in hypomyelinated brain may reveal why deletion of the myelin basic protein gene leads to subcortical seizure patterns. Analysis of these models offers insight into developmental processes that control the cellular expression and plasticity of Na+ channel genes, and will help to clarify mechanisms of hereditary Na+ channel-based epileptogenesis.

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©2001 The Novartis Foundation

Beta subunits: players in neuronal hyperexcitability?

Lori L. Isom

Voltage-gated Na+ channels are glycoprotein complexes responsible for initiation and propagation of action potentials in excitable cells such as central and peripheral neurons, cardiac and skeletal muscle myocytes, and neuroendocrine cells. Mammalian Na+ channels are heterotrimers, composed of a central, pore-forming a subunit and two auxiliary b subunits. The a subunits form a gene family with at least 10 members. Mutations in a subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome, and hyperkalaemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode Na+ channel b subunits with at least one alternative splice product. A mutation in the b1 subunit gene has been linked to generalized epilepsy with febrile seizures plus type 1 (GEFS+1) in a human family with this disease. Na+ channel b subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. More recently, they have been shown to function as cell adhesion molecules in terms of interaction with extracellular matrix, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. Structure-function studies have resulted in the preliminary assignment of functional domains in the b1 subunit. A Na+ channel signalling complex is proposed that involves b subunits as channel modulators as well as cell adhesion molecules, other cell adhesion molecules such as neurofascin and contactin, RPTPb, and extracellular matrix molecules such as tenascin..

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©2001 The Novartis Foundation

Modulation of sodium channels in primary afferent neurons

Stuart Bevan and Nina Storey

Electrophysiological studies have revealed that the properties of voltage-gated Na+ channels can be modified by phosphorylation. Na+ channels have multiple sites for phosphorylation by protein kinases A and C (PKA and PKC). A change in the phosphorylation state of Na+ channels is an important mechanism of neuromodulation for both central and peripheral neurons. In isolated primary afferent sensory neurons, application of an inflammatory mediator, prostaglandin E2 (PGE2), causes an increase in excitability associated with a hyperpolarizing shift in the activation curve of the tetrodotoxin-resistant (TTX-R) Na+ currents. The experimental evidence indicates that the effect of PGE2 is mediated by an elevation in cAMP levels and activation of PKA. This potentiation of TTX-R Na+ channel activity is in marked contrast to the inhibitory effects of PKA and PKC on tetrodotoxin-sensitive (TTX-S) currents in central neurons. Infection of dorsal root ganglion neurons with Herpes simplex virus (HSV) results in an abolition of excitability associated with a selective loss of both TTX-S and TTX-R Na+ currents: voltage-gated Ca2+ and K+ channels are unaffected by HSV infection. The loss of Na+ current is due to a virally induced internalization process and requires extracellular Na+.

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©2001 The Novartis Foundation

Sodium channels in primary sensory neurons: relationship to pain states

John N. Wood, Armen N. Akopian, Mark Baker, Yanning Ding, Fleur Geoghegan, Mohammed Nassar, Misbah Malik-Hall, Kenji Okuse, Louisa Poon, Samantha Ravenall, Madhu Sukumaran and Veronika Souslova

Electrophysiological studies of dorsal root ganglion (DRG) neurons, and the results of PCR, Northern blot and in situ hybridization analyses have demonstrated the molecular diversity of Na+ channels that operate in sensory neurons. Several subtypes of a-subunit have been detected in DRG neurons and transcripts encoding all three ß-subunits are also present. Interestingly, one a subunit, Nav1.8, is selectively expressed in C-fibre and Ad fibre associated sensory neurons that are predominantly involved in damage sensing. Another channel, Nav1.3, is selectively up regulated in a variety of models of neuropathic pain. In this review we focus on Na+ channels that are selectively expressed in DRG neurons as potential analgesic drug targets. In the absence of subtype specific inhibitors, the production of null mutant mice provides useful information on the specialized functions of particular Na+ channels. A refinement of this approach is to delete Na+ channel genes flanked by lox-P sites in the sensory ganglia of adult animals, using viruses to deliver the bacteriophage Cre recombinase enzyme.

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©2001 The Novartis Foundation

Sodium channels and epilepsy electrophysiology

Michael M. Segal

We examined the electrophysiology of epilepsy in the simplest system that exhibits epileptiform activity: microisland cultures that contain only one neuron. Some of these solitary excitatory hippocampal neurons generate the 'ictal' epileptiform activity characteristic of seizures. These neurons have endogenous (non-transmitter-mediated) bursts of activity that last for many seconds and appear to be driven by a persistent Na+ current. We examined this persistent Na+ current at the single channel level by recording the late openings of Na+ channels using outside-out patch recordings. Phenytoin reduced the probability of these late channel openings, but had less effect on the early channel openings that make up the peak Na+ current. The reduction of late channel openings was larger with pulses to more depolarized voltages. In contrast, the effect on early channel openings was similar at all voltages. There was little effect of phenytoin on the duration of channel openings and no effect on open channel current. This suggests that the persistent Na+ current is crucial in generating seizures. A good strategy for selecting anticonvulsants may be to search for drugs that more selectively block the persistent Na+ current at depolarized voltages. Such drugs could combine effectiveness and reduced side effects.

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©2001 The Novartis Foundation

Therapeutic concentrations of local anaesthetics unveil the potential role of sodium channels in neuropathic pain

Gary R. Strichartz, Zhongren Zhou, Catherine Sinnott and Alla Khodorova

Neuropathic pain is frequently associated with hyperexcitability of primary afferents, characterized by spontaneous impulses and repetitive firing. Electrophysiology and molecular biology reveal changes in dorsal root ganglion Na+ channels under conditions of neuropathic pain, but the manner by which these changes alter the physiology of sensory afferents remains unknown. Equally mysterious is the mechanism by which i.v. local anaesthetic-like Na+ channel blockers suppress neuropathic pain behaviour at concentrations well below those reported for channel inhibition. We have compared the anti-allodynic actions of i.v. lidocaine (L) and stereoisomers of mexiletine (R-M, S-M), in rats after spinal nerve ligation, with their ability: (1) to inhibit fast, tetrodotoxin-sensitive neuronal Na+ currents, elicited by brief (1 ms) pulses, at 10 Hz, from 'resting' potentials (-80, -60 mV) and (2) to suppress the seconds long plateau and the repetitive firing produced in axons by slowing of Na+ channel inactivation (e.g. using scorpion a-toxins). Both L and R-M at 5-10 mM relieved allodynia; S-M was ineffective. Na+ currents also were inhibited by M, with affinities that were increased by both repetitive 'firing' (KR,S = 5 mM) and depolarization of the 'resting' membrane (KR = 15 mM; KS = 30 mM). Stereopotency ratios depended on the manner in which different states of the channel were inducted. Both L and M shortened the action potential's 'plateau' in a-toxin treated axons, without reducing the spike, and suppressed repetitive firing with IC50s "5 mM, and no stereoselectivity. These findings together demonstrate that Na+ channel blockers, at 'therapeutic' concentrations, can inhibit neuronal hyperexcitability.

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©2001 The Novartis Foundation

Molecular mechanisms of gating and drug block of sodium channels

William A. Catterall

Voltage-gated Na+ channels are composed of an a subunit of 260 kDa associated with ß subunits of 33-36 kDa. a subunits have four homologous domains (I through IV) containing six transmembrane a helices (S1-S6). The S4 segments serve as voltage sensors and move outward to initiate activation. The S5 and S6 segments and the short membrane-associated loops between them form the pore. Fast inactivation is mediated by closure of an inactivation gate formed by the intracellular loop between domains III and IV. The 3-D structure of the inactivation gate has been determined by NMR spectroscopy, revealing the conformation of the pore-blocking IFM motif. Peptide scorpion toxins that alter gating of Na+ channels bind to the extracellular ends of the IIS4 and IVS4 segments, trap them in either an activated or non-activated position, and thereby selectively alter channel activation or inactivation. Voltage sensor-trapping may be a general mechanism of toxin action on voltage-gated ion channels. Local anaesthetics block the pore of Na+ channels by binding to a receptor site in segment S6 in domains III and IV. Anticonvulsants and antiarrhythmic drugs also interact with this site. A high-affinity Na+ channel blocker has recently been developed with this site as its target. The emerging knowledge of the molecular mechanisms of Na+ channel gating and drug block may allow development of novel therapeutics for epilepsy, cardiac arrhythmia and persistent pain syndromes.

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©2001 The Novartis Foundation