Multiple Sclerosis and Related Disorders (2013) 2, 270–280

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/msard

REVIEW

Potential therapeutic mechanism of K + channel block for MS Mark D. Bakern Neuroscience and Trauma, Barts and the London School of Medicine, Queen Mary University of London, Blizard Institute, 4 Newark Street, Whitechapel, London E1 2AT, UK Received 1 November 2012; received in revised form 7 January 2013; accepted 20 January 2013

KEYWORDS

Abstract

K + channel; 4-aminopyridine; Axon; Multiple sclerosis; Potassium channel; Demyelination

While the potential use of K + channel blockers in MS has been explored over many years, the approval in the US, and more recently in the UK, of fampyra (fampridine, 4-aminopyridine, 4-AP) as a symptomatic treatment for walking disability, has reawakened interest. Recent years have seen a real improvement in the treatment options for relapsing remitting MS, but the disease remains inadequately treated, with the progressive phase (characterised by irreversible functional loss) lacking any effective therapy. Whether the symptomatic relief afforded by 4-AP translates into neuroprotection, remains poorly investigated, although there is no clear reason why this would be expected. Importantly, future clinical studies may shed light on this question. This review includes an overview of axonal K + channel expression and pharmacology, and the logic of the use of K + channel blockers derived from observations in experimental studies of demyelination and synaptic transmission. It provides an insight into the probable biophysical actions of 4-AP, and how its action may aid in the symptomatic treatment of MS. The key message of this review is that 4-AP is a blocker of neuronal K + channels, and its administration is known to be of value in the symptomatic treatment of some patients. The details of the mechanism underlying the beneficial effects remain somewhat vague, and the molecular target has not been properly defined. The useful mechanism is likely to include an action on synaptic function, but whether it is the presynaptic terminal or the presynaptic axon that is the primary target is unknown. It is argued that because of the apparent inability of 4-AP to increase safety factor in experimental demyelination when clinically relevant concentrations are used, it cannot be the ideal pharmacological agent for treating demyelination by the widening of axonal action potentials. That said, it remains a possibility that the useful therapeutic effect of 4-AP may involve subtle changes in axonal excitability mediated by a selective K + channel block, exploiting a naturally occurring redundancy of synaptic function. & 2013 Elsevier B.V. All rights reserved.

n

Tel.: +44 (0)207 882 2287. E-mail address: [email protected]

2211-0348/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msard.2013.01.005

Potential therapeutic mechanism of K + channel block for MS

271

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of K + channels in axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Distribution and function of K + channels in peripheral myelinated axons . . . . . . . . . . . . . . . . . . . . . 2.2. Molecular identity of GKf and GKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Pharmacology of GKs indicates equivalence with IM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Comparison of CNS and PNS axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The function of myelin in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Normal K + channel distribution in CNS axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. K + channelopathies affecting CNS and PNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. The functional effects of demyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. K + channel block and symptomatic treatment of demyelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Origin of K + channel strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Clinical findings on walking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Can K + channel block contribute to the useful effects at therapeutic 4-AP concentrations? . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

Introduction

Our basic understanding of the distribution and function of K + channels in mammalian axons comes primarily from electrophysiological and pharmacological experiments on rodent and amphibian peripheral nerve, although this has recently been supplemented by non-invasive recordings from peripheral nerves of patients with K + channelopathies. There are some important differences between peripheral and central axons, and this will be addressed below. 4aminopyridine (4-AP) has been used both experimentally and clinically as a blocker of axonal K + channels for many years, and this review argues that a detailed understanding of the functioning of axonal and pre-synaptic K + channels may yet prove necessary in elucidating the compound’s clinically useful properties. The clinical utility of 4-AP, and its effects on walking, are dealt with in more detail in an accompanying paper by Hadavi et al. (2013).

2.

Roles of K + channels in axons

2.1. Distribution and function of K + channels in peripheral myelinated axons Dubois (1981) presented evidence for the presence of kinetically fast and kinetically slow K + currents in frog nodes of Ranvier, called IKf and IKs, respectively, by analysing tail currents in voltage-clamp experiments. Subsequently it was discovered that mammalian nerve also generates similar K + currents, but in healthy adult nerve, the kinetic characteristics of a propagated action potential depend very little on voltage-gated K + channel activity. The accommodative properties and repetitiousness of axons are, however, known to be dependent upon these two major classes of K + channel, generating kinetically fast and slow currents (Dubois, 1981; Baker et al., 1987; R¨ oper and Schwarz, 1989; Safronov et al., 1993), that can be distinguished

271 271 271 272 273 273 273 273 274 274 275 275 277 277 278 278 278 278

pharmacologically by sensitivity to 4-AP and tetraethylammonium ions (TEA), respectively. The work of Chiu et al. (1979), in which rabbit nodal currents were discovered to lack the delayed-rectifier found at frog nodes, was early evidence for a complementary distribution of Na + channels and kinetically fast K + channels (GKf; where G indicates conductance), along mammalian myelinated nerve, rather than a co-localization. Electrophysiological experiments detailing the functional distribution of K + channels include the recording of electrotonus in rat spinal root axons (Baker et al., 1987), where such recordings can be understood in accordance with the Barrett and Barrett model of the axon (Barrett and Barrett, 1982, reviewed by e.g. Baker (2000). The electrotonic waveform produced by passing a rectangular constant-current depolarizing pulse into a spinal root, anaesthetized by prior exposure to tetrodotoxin (TTX), comprises fast and slow components. The fast component is caused by the potential difference generated across the nodal and myelin resistances in parallel; with a time-constant determined by the myelin resistance and capacitance, and the parallel resistance and capacitance of the node. The slow component reflects the much slower charging of the internodal axolemma through the myelin resistance. Both fast and slow components of electrotonus are modified by K + channel activation, whose effects can be selectively eliminated by applying TEA and 4-AP (Fig. 1). The effect of TEA on the potential response to brief and repeated depolarizations underlined the conclusion that kinetically slow K + channels (GKs), were expressed at the nodes (Baker et al., 1987). The response to 4-AP was primarily (although not exclusively) to enhance slow electrotonus in the depolarizing direction, consistent with the blockade of GKf, expressed in the internodes (although there was evidence for a small amount of classical delayed rectifier, 4-AP sensitive current, at nodes in both sensory and motor axons). Consistent with these findings, 4-AP, but not TEA, enhanced the depolarizing afterpotential recorded intra-axonally in single axons (Fig. 2), but TEA, not 4-AP, depolarized axons at rest. These findings indicate physiological roles for GKf in the control of depolarizing afterpotential

272

M.D. Baker

Fig. 1 (A and B) TEA ions abolish the sag in depolarizing electrotonus in a ventral root subjected to depolarizing and hyperpolarizing constant currents. (A) Before application of TEA, in the presence of TTX and 5 mM 4-AP. (B) After additional application of 5 mM TEA. Sag in the waveform affects the fast phase (on this time scale the almost instantaneous step at the beginning and end of the applied current). The suppression is prevented in (B), consistent with block of TEA sensitive K + channels operating at the nodes. (C and D) 4-AP enhances the amplitude of slow electrotonus, particularly, although not exclusively, in the depolarizing direction. (C) Dorsal root electrotonus recorded in the presence of TTX and 5 mM TEA. D, additional application of 5 mM 4-AP increases the slow component, while fast electrotonus is unchanged, indicative of block of internodal K + channels. From Baker et al. (1987), with permission.

Fig. 2 Depolarizing afterpotentials (DAPs) recorded intracellularly from a motor axon in a ventral root. The peak of the propagated action potential has been truncated in each trace. Where the resting membrane potential is held close to 60 mV, exposure to TEA ions does not increase the DAP, whereas the addition of 4-AP substantially increased its amplitude, consistent with the effect of 4-AP on slow electrotonus and the block of internodal K + channels. From Baker et al. (1987), with permission.

(DAP) amplitude and duration, and for a GKs contribution to resting membrane potential and very strongly to accommodation.

2.2.

Molecular identity of GKf and GKs

We now know that GKf and GKs include the molecular entities KV1.1/KV1.2 and KV7 subtypes (including KCNQ2 and 3), respectively (e.g. Rasband et al., 1998; Corrette et al., 1991; Ulzheimer et al., 2004; Devaux et al., 2002; Devaux et al., 2004; Arroyo et al., 2002), although there is evidence provided by single-channel recording from human axons that actually 5 different functional classes of voltagegated K + channel exist (Reid et al., 1999), including I, S and F channels previously described in amphibian and rat axons, whose overlapping characteristics give rise to the macrosopic

voltage-dependent currents. The I channel, that contributed the majority of current recorded in large patches of internodal membrane in human nerve, was blocked by 1 mM a-dendrotoxin (a-DTX), and is therefore a probable functional correlate of the molecular entities KV1.1 and KV1.2 (reviewed by Harvey, 2001), although perhaps not the only one, as other channels with different characteristics were also blocked by a DTX (Reid et al., 1999). The voltagegated K + channel KV3.1b has also been immunohistochemically associated with some central mammalian nodes (Devaux et al., 2003), and an additional Na + dependent K + channel has been described at amphibian peripheral nodes (Koh et al., 1994). The functional effects of 4-AP and the immunohistochemically located juxtaparanodal expression of KV1.1 and KV1.2, strongly support the conclusion that mammalian GKf includes these K + channels (Wang et al., 1993; Rasband

Potential therapeutic mechanism of K + channel block for MS

273

Fig. 3 XE991 blocks the slow K + current in an isolated node current-clamp. (A: a and b) Action potentials recorded in currentclamped single node of Ranvier, before and after exposure to XE991 (100 mM). XE991 is a blocker of the M-current, and changes the accommodative properties of the rat node, encouraging repetitive firing to a prolonged depolarization (B: a and b), while having a minimal effect on action potential current-threshold and action potential duration. From Schwarz et al. (2006), with permission.

et al., 1999; reviewed by Rasband and Trimmer, 2001). Further proof of the functional importance of the fast delayer rectifier current provided by these K + channels in normal axons is the evidence for hyperexcitability in the CA3 mossy fibre axons, and for the cold-induced neuromyotonia found in KV1.1 knock-out mice (Smart et al., 1998), the latter thought to result from instability in the membrane potential near the motor nerve ending where action potentials are already prolonged by the effect of cooling on Na + channel gating kinetics.

2.3. Pharmacology of GKs indicates equivalence with IM While the channels underlying GKf are serendipitously targeted by toxins from Mamba species, for many years the molecular identity of GKs remained shrouded in mystery, and its pharmacology defined by block from the outside by high concentrations of TEA and Ba2 + ions. A clue provided by a study of a mutation in KCNQ2 that was associated with myokymia and neonatal epilepsy (Dedek et al., 2001), led to the discovery that KCNQ2 is a nodal and initial segment channel that could fulfil the role of GKs, exhibiting slow gating kinetics and the correct insensitivity to 4-AP (Devaux et al., 2004). The pharmacology of the slow axonal current is consistent with it being KCNQ2 and 3 (KV7.2 and KV7.3 channels), equivalent to the pyramidal neuron G-protein pathway regulated K + channel IM, that is inhibited by acetylcholine (e.g. Wang et al., 1998; Shah et al., 2008). The axonal current is targeted by molecules either blocking (e.g. XE991) or activating (e.g. retigabine) the M-current (Schwarz et al., 2006), Fig. 3, reviewed by Brown and Passmore (2009).

3. 3.1.

Comparison of CNS and PNS axons The function of myelin in the CNS

Central myelinated axons differ from peripheral myelinated axons in important ways. Schwann cells sequentially myelinate a single peripheral axon, whereas oligodendrocytes provide

myelin for several neighbouring central axons, and astrocyte processes closely associate with central nodes (reviewed by Poliak and Peles, 2003). Sheath structural integrity in the CNS and PNS is maintained by different proteins that also provide tissue specific immunogenic epitopes, used as an explanation for the selective autoimmune destruction of central myelin in MS (reviewed by Sospedra and Martin, 2005). It is widely understood that the provision of a myelin sheath around an axon substantially reduces the effective electrical capacity of the structure, allowing rapid and energy efficient impulse conduction, where only the nodes of Ranvier are actively involved in impulse generation. The location of Na + channels in the axolemma at nodes of Ranvier confers excitability, but the very low density of Na + channels in the internodal axolemma (under the myelin) means that this region of the axon membrane is inexcitable, and the acute loss of myelin thus gives rise to conduction failure. The ability of myelin to speed impulse conduction has been known for many years to be dependent upon the axon diameter (Rushton, 1951), and the impact on conduction velocity diminishes in smaller axons, owing to the necessity of having nodes of Ranvier strung closer together in order to maintain intact conduction. In fact, in the periphery, axons of 1 mm diameter or smaller are not myelinated, but this is not true of the CNS, where the vast majority of axons have sub-micron diameters ( for example in the optic nerve commonly 0.2 mm) and yet are thinly myelinated (Hildebrand and Mohseni, 2005; Perge et al., 2009), strongly suggesting that myelination plays other vital roles in the CNS. These probably include reducing the energy required for impulse transmission (by reducing the effective electrical capacity of the axons), and also preventing axonal ephaptic cross-talk (by confining excitability to nodes).

3.2.

Normal K + channel distribution in CNS axons

The distribution of 4-AP sensitive K + channels in adult CNS axons appears to be similar to that proposed for PNS axons, and some of the most persuasive evidence for this has been

274

M.D. Baker

Fig. 4 Left-hand panel. Demonstration of the complementary distribution of Na + channels (green), and 4-AP sensitive K + channels, KV1.2, (blue) in optic nerve axons, revealed by immunohistochemistry. The cell adhesion molecule caspar (contactin associated protein) is present at the region of apposition of the terminal loops of myelin (axoglial junctions) in the paranode (red). Calibration bar 10 mm. From Rasband and Shrager 2000 with permission. Right-hand panel. Staining for KV1.2 (green) and KCNQ2 (red) in spinal cord white matter axons shows that KCNQ2 is found in highest density at nodes of Ranvier, and does not co-localize with KV1.2, which is juxta-paranodal and internodal. Calibration bar is 10 mm. From Devaux et al. (2004), with permission.

provided by exquisite immunocytochemical studies, where the distribution of Shaker homologue K + channels is revealed by selective fluorescently labelled antibodies to be complementary with that of Na + channels (e.g. Rasband and Shrager, 2000; Devaux et al., 2004). The Na + channels are located at the nodes, and the 4-AP sensitive K + channels under the myelin in the internodal membrane, Fig. 4. However, there is also evidence for other 4-AP sensitive K + channels, apparently present at central nodes of Ranvier in adult rodents, whose blockade with 4-AP at a high concentration (500 mm) gives rise to substantial action potential widening (Devaux et al., 2002). In juvenile animals, channels sensitive to the natural polypeptide toxins dendrotoxin-I (DTX-I) and kaliotoxin (KTX) (and also 4-AP) are present at nodes (likely to be KV1.1 and KV1.2 for DTX-I, and KV1.1 for KTX; Dolly and Parcej (1996 and Mourre et al. (1999), but with maturation, this toxin sensitivity largely or completely disappears, consistent with the polypeptide toxins being unable to access the channels and interpreted as sequestration of sensitive K + channels to the juxta-paranodes and internodes during development (Devaux et al., 2002). Nevertheless, a 4-AP sensitivity remains, even when myelination is complete, that is not accounted for by a different 4-AP sensitive K + channel, also expressed at nodes in central axons, KV3.1b (Devaux et al., 2003), and this finding remains unexplained. There is functional evidence for kinetically slow K + channel expression in mature optic nerve axons, similar to those found in the periphery (i.e. GKs), and these channels are responsible for post-activity hyperpolarization (Gordon et al., 1988; Eng et al., 1988). Because the channels are accessible to TEA ions, they are thought to have a nodal expression pattern, and correspond well with the localization of KCNQ2/KCNQ3, visualized by selective fluorescent antibodies in spinal cord sections (Devaux et al., 2004), Fig. 4, right-hand panel.

3.3.

K + channelopathies affecting CNS and PNS

The role of KV1.1 channels in human CNS and PNS is indicated by the genetic channelopathy episodic ataxia type

1 (EA1), due to loss-of-function mutations in KV1.1 (Zuberi et al., 1999). Centrally this K + channel dysfunction produces sporadic attacks of discoordination, originating in the cerebellum, usually triggered by emotion or stress, while peripherally it is associated with myokymia, due to spontaneous activity in motor axons. Threshold electrotonus, a technique for recording electrotonus in axons indirectly by non-invasive excitability measurements (Bostock and Baker, 1988) reveals abnormalities in EA1 patients similar to those produced in rat axons in vitro by 4-AP (Tomlinson et al., 2010). Similarly, the role of KV7.2 channels in human CNS and PNS is indicated by mutations in the KCNQ2 gene for the slow KV7.2 channels. In the CNS this causes benign familial neonatal epilepsy (BFNE), which is usually evident within a week of birth, but resolves within a few months, although an increased susceptibility to seizures may remain for life. In this case the changes in peripheral nerve excitability, analogous to those produced in vitro by TEA or XE991, can be revealed by threshold electrotonus although they are normally asymptomatic (Fig. 5; Tomlinson et al., 2012).

3.4.

The functional effects of demyelination

Acute segmental demyelination gives rise to conduction failure by short-circuiting local circuit currents, thus preventing impulse propagation through the damaged region. The reasons for the failure include an increase in the effective capacity of the internode, as in this circumstance axonal internodal membrane becomes a capacitative load on the last working node of Ranvier. Also, the uncovering of kinetically fast K + channels in the internodal membrane will tend to work against the spread of depolarization from the last working node. The axonal membrane potential is therefore locally stabilized, in part by internodally expressed K + channels. Such complete failure of conduction is one mechanism for ‘negative’ symptoms associated with demyelination, although intermittent and activity-dependent conduction failure may also occur (e.g. Bostock and Grafe, 1985). However, axons can recover the capacity to conduct through the affected region by the formation of a new distribution of Na + channels, with the

Potential therapeutic mechanism of K + channel block for MS

275

Fig. 5 Corresponding effects of K + channel loss of function mutation on threshold-electrotonus and the selective block of K + channels on electrotonus. Panel A, loss of function mutation in KV7.2 (KCNQ2) (associated with benign familial neonatal epilepsy, BFNE), reduces the sag in excitability associated with depolarizing electrotonus in motor nerve, and the post-depolarization suppression of excitability. Loss of function in KV1.1 (associated with episodic ataxia type-1, EA1), enhances the amplitude of threshold-electrotonus, consistent with an increase in internodal membrane resistance. Panel B, the effects of selective blockade of GKs and GKf with TEA and 4-AP, respectively, on rat peripheral nerve electrotonus. TEA and 4-AP cause a block of the hyperpolarizing sag, and an increase in the amplitude of depolarizing electrotonus, respectively. Figure provided by Hugh Bostock.

conferment of excitability to previously inexcitable internodal membrane (Bostock and Sears, 1978), although the impulse conducts with a reduced velocity. This finding has been taken as the explanation of the persistent slowing of impulse progression in patients found in evoked potential studies (e.g. Halliday et al., 1973; Asselman et al., 1975). The continuously propagated impulse is terminated by the activation of K + channels expressed in the previously internodal membrane, and the block of these channels using 4-AP is known to widen the action potential, and give it an increased duration (Sherratt et al., 1980). Demyelination is associated not only with ‘negative’ symptoms, but also with ‘positive’ where the positive symptoms (such as paraesthesias and neuromyotonia) have been explained by the generation of ectopic activity in demyelinated axons, and in the emergence of mechanosensitivity in affected fibre tracts (e.g. Smith and McDonald, 1980; Calvin et al., 1982; Nordin et al., 1984; Baker and Bostock, 1992; Baker, 2000). In attempting to understand the origin of ectopic discharges from demyelinated peripheral axons, Hugh Bostock and I found it necessary to propose the presence of a persistent Na + current, capable of providing a pacemaker potential at an ectopic site (Baker and Bostock, 1992; Baker, 2005). Such a current operates within a range of sub-threshold membrane potentials, in fact within the range of negative membrane potentials that may be importantly linked to the useful activity of 4-AP, elucidated in arguments presented below. We found that exposure to small amounts of 4-AP (where the effective concentration was not well quantified, although sub

millimolar), were useful in promoting experimental ectopic discharge in our studies, almost certainly because the blocker increased axonal membrane excitability.

4. K + channel block and symptomatic treatment of demyelination 4.1.

Origin of K + channel strategy

The logic of this approach derives from initial observations by Uhthoff in the 19th century (reviewed by Smith and Waxman, 2005), that changes in body temperature can affect symptom severity in MS. There is evidence that symptoms can worsen with warming through apparently quite benign activities such as showering, sunbathing or exercise, and symptoms can be transiently beneficially affected by cooling (e.g. Scherokman et al., 1985). The failure of action potentials to propagate continuously through demyelinated regions is known to show an acute temperature dependence, where cooling increases conduction safety factor, and warming reduces it (e.g. Smith et al., 2000). This is explained by an effect of temperature on the axonal Na + currents, where raising the temperature makes Na + currents briefer, and action potentials shorter, until at some critical degree of warming, insufficient depolarizing local circuit current flows, and conduction fails. One possible way to enhance conduction and increase safety factor was therefore to increase the duration of the action potential, and this could be done pharmacologically by blocking voltage-gated K + channels.

276

M.D. Baker

Fig. 6 Top section; (A) EPSP recorded from spinal motorneurone increases in amplitude after systemic administration of 4-AP (IV, 1 mg/kg). The increase in EPSP amplitude is accounted for by a change in the probability of individual boutons operating in response to afferent activation. Amplitude of components generated by the activation of whole number of boutons and their probability of occurance shown at times after administration in (B), (C) and (D), revealed using the mathematical technique of deconvolution. The results show that the probability of individual synaptic bouton activation is increased, resulting in more common large EPSP events when individual boutons operate simultaneously (from Jack et al., 1981, with permission). Lower section; an interpretation of the result implies synaptic redundancy in the nervous system. In this example, only two boutons operate when an afferent impulse arrives before 4-AP (left hand panel), but after exposure to the drug, three boutons operate simultaneously, activating the post-synaptic neuron (right-hand panel).

Hugh Bostock proved that this strategy might work by demonstrating the beneficial effects of K + channel blockers on the temperature at which impulse transmission failed (Bostock et al. 1981), where a critical temperature of 35 1C in previously demyelinated axons, increased to 40 1C in the presence of K + channel blockers. The idea that beneficial clinical effects might be achieved by selectively blocking the K + channels expressed in internodal axon membrane is well founded on classical experimental studies (e.g. Bostock et al., 1978; Sherratt et al., 1980), but the concentrations of blockers used experimentally are far in excess of those found to be clinically useful. So paradoxically, the useful action of 4-AP may not involve action potential widening at all, and a corollary of this is that an agent that effectively

widens nerve action potentials may not yet have been tested in the clinic. Clinical experimental findings on 4-AP have accumulated over many years suggesting that K + channel blockade in MS can improve visual-field defects and reduce fatigue (e.g. Davis et al., 1990; Rossini et al., 2001), although, presumably because the agent does not discriminate well between K + channel sub-types, higher doses are associated with side effects. Whether K + channel blockade-dependent improvements in impulse transmission can lead to enhanced survival of axons that have been in close proximity to autoimmune attack, remains an open question, although in the case of MS with further use of fampyra in man, more evidence on this might become available.

Potential therapeutic mechanism of K + channel block for MS

4.2.

Clinical findings on walking

In an accompanying review, Hadavi et al. (2013) have summarized recent clinical findings for fampridine efficacy and safety in a phase II trial, and two phase III trials investigating improvement in walking speed and patients’ subjective perception of their disabilities. In brief, it seems that low dose fampridine benefits a fraction of patients, is associated with a meaningful increase in walking speed, although also exhibiting a variety of potential adverse effects. The available evidence indicates no obvious increase in the rate of falling in patients given the drug, in comparison to placebo.

4.3. Can K + channel block contribute to the useful effects at therapeutic 4-AP concentrations? The free CSF concentration in patients receiving the drug is expected to be at most within the low single mM range (with a blood plasma concentration exceeding 60 ng/ml for evident therapeutic effects (analytical specification, National Medical Services Inc; http://toxwiki.wikispaces.com/file/view/Dalfam pridine.pdf)). This is a considerably lower concentration than that used to widen action potentials and because the literature also points to an apparently separate effect of 4-AP on synapses, this has led to the suggestion that the primary target cannot be demyelinated axons, but rather central and or peripheral synapses (Smith et al., 2000). However, this view is not monolithic, as there is also evidence for an effect of 4-AP within the single mM range, on enhancing axonal responses propagated across a circumscribed injury in spinal-cord (Shi and Blight, 1997; Shi et al., 1997), where injury per-se is suggested to enhance the effect of the drug. The mechanism and consequences of post-traumatic demyelination have not been well defined, although the change in susceptibility to 4-AP is conceivably related to an altered role of 4-AP sensitive K + channels at damaged nodes. Thus, these findings may be another expression of the effect of low concentration 4-AP on axonal excitability, reported more recently in undamaged cerebellar parallel fibres (discussed below), but in this case possibly aiding nodal recruitment during impulse propagation. One piece of evidence clearly indicating that an action on some aspect of synaptic function must occur within the clinically achievable concentration range, is that the Hreflex resulting from stimulation of sciatic Ia afferents is enhanced in rat following systemic dosing with 4-AP (Smith et al., 2000). These data were consistent with those findings reported much earlier by Julian Jack and colleagues, where an action of systemically administered 4-AP gave rise to changes in the behaviour of Ia afferent synapses onto motoneurones, substantially altering their recruitment probability, and even turning-on previously silent boutons (Jack et al., 1981), Fig. 6. It is uncertain why such a synaptic effect might be useful in patients with mobility issues, although it is possible to conceive of a raised level of synaptic drive in the spinal cord, increasing motorneurone excitability and helping remaining uninterrupted descending motor tracts to activate motor units. However, it is still not possible to rule out a K + channel involvement in altered nervous system function when exposed to low therapeutic concentrations of 4-AP, and this

277 is because K + channels do more than control the shape of an action potential. Voltage-gated K + channels also control membrane excitability and determine impulse firing patterns. The control of action potential width and excitability, that may at first sight seem inextricably linked, is undertaken in mammalian neurons by specialized and different sub-types of K + channel (reviewed by Johnston et al., 2010). The firing threshold of a neuron is controlled by low voltage-activated K + channels, i.e. those opened by depolarizations close to the normal resting potential, and this includes KV1.1 (e.g. Baker et al., 2011), and KV7 (KCNQ, GKs). Remarkably, removing KV1.1 function in primary sensory neurons has a clear functional effect on threshold, but leaves action potential width unaffected (Baker et al., 2011). Other channels, including the KV3 and KV2 subfamilies, are activated in response to larger depolarizations, and underlie the currents used to repolarize and terminate action potentials (Johnston et al., 2010). Admittedly, given the expression of a limited set of K + channel subtypes and the normally circumscribed distributions of expression, a similar association of channel subtype and function for axons may be invalid. In other words, where the local K + conductance over a wide range of membrane potentials is dominated by channels incorporating, for example, KV1.1, then the same channel may have roles in both controlling action potential shape and in controlling threshold. This view would fit with the findings on the functional effects of DTX-I and KTX on juvenile central axons (Devaux et al., 2002) for nodally expressed KV1 channels, that must contribute to controlling action potential width. Nevertheless, even supposing a completely uniform population of channels, the reported voltage-dependence of 4-AP block of K + channels (where block is greater at more negative membrane potentials, e.g. Howe and Ritchie, 1991), would tend to result in a predominant effect on membrane excitability. This is because those channels operating near the resting potential would be predisposed to block by the drug. It therefore remains a possibility that 4-AP sensitive channels, including KV1.1, are particularly sensitive to the drug at negative membrane potentials and mediate the therapeutic effect by subtly increasing membrane excitability. That said, an axonal target could only be compatible with the effect on synapses if there was a change in excitability at central nodes of Ranvier, where 4-AP sensitive K + channels had a nodal and pre-synapse representation and their block could therefore affect nodal or pre-synapse excitability, or the excitability of critical axon collateral branch points. However, evidence that such an effect on axonal excitability in fact already exists for normal parallel fibres in the cerebellum when exposed to low, single mM concentrations of 4-AP (Alvin ˜a and Khodakhah, 2010),implying that 4-AP sensitive K + channels must be at central nodes and contributing to the control of excitability. In these experiments, the authors concluded that low mM concentrations of 4-AP did not act to change the probability of transmitter release at parallel fibre-Purkinje neuron synapses (because of the lack of effects on paired-pulse facilitation), but it probably increased the responsiveness of the axons to the locally applied electrical stimulation by altering their excitability. That 4-AP sensitive K + channels may be involved in controlling pre-synaptic axon excitability is also supported by their distribution of expression. KV1.1 and

278

M.D. Baker

Fig. 7 A cartoon central node action potential is shown, before and after the application of 4-AP. The effects of low-concentrations of the drug are typified by raised excitability and a reduced induction time, caused by block of KV1 sub-family K + channels at sites at which they contribute to resting membrane conductance. Higher concentrations of the drug are proposed to widen the action potential, and eliminate an after-hyperpolarization generated by the activation of high-voltage-gated K + channels.

KV1.2 are expressed in the pre-synapse, and at locations in presynaptic axons that may regulate action potential invasion of terminals (Wang et al., 1993, 1994). Fig. 7 attempts to simplify the proposed actions of 4-AP on voltage-gated K + channels at either normal or widened central nodes, or at pre-synaptic terminals. Finally, other lines of evidence exist in the literature, implicating different molecular mechanisms for 4-AP. It has been suggested that 4-AP may act to facilitate vesicle exocytosis at the pre-synapse (Thesleff, 1980), or to enhance Ca2 + currents. There is evidence from voltageclamp studies carried out in neurons, that high concentrations of 4-AP increase the size of barium enhanced HVA Ca2 + currents (Wu et al., 2009), 100 mM (the lowest concentration reported) significantly increased the Ca2 + current. In spinal cord slices, and in diaphragm muscle fibres, 500 mM 4AP increased synaptic currents and prolonged synaptic potentials in a manner apparently independent of K + channels, and consistent with an action on pre-synaptic Ca2 + channels (Wu et al., 2009). However, taking a very liberal view it seems likely that concentrations of 50 mM or greater may be required to generate these effects, making them less likely to be clinically relevant.

5.

Conclusion

Myelinated axons express a variety of K + channel subtypes that can be characterized by their distribution, function and pharmacology. 1. The channels residing in large part underneath the myelin in a mature nerve, belonging to the sub-family KV1, are blocked by 4-AP, but so too are KV3 channels, thought to be expressed at central nodes. 2. 4-AP is of known utility in treating walking disability in MS with one-third to one-half of patients seeing improvements. 3. While it seems unlikely that the drug could produce a lengthening of action potentials in demyelinated axons in

the clinical setting, because the available concentration is too low, the drug may act to change axonal membrane excitability and in this way impact synaptic drive in the spinal cord. It is possible that the drug may do this for two reasons. It is expected to selectively interact both on the basis of the molecular structure of K + channels, and also because it preferentially targets K + channels operating at negative membrane potentials. 4. Convincing evidence is available that following afferent activity, the probability of individual pre-synaptic bouton activation in the cord is less than one, and this is increased by administration of systemic 4-AP, through a mechanism that has not yet been fully defined, but that may include an action on K + channels. 5. In the light of the therapeutic benefits in some patients, more fundamental work on the molecular mechanisms underpinning the useful effects of 4-AP is justified.

Conflict of interest I have acted as a consultant to Biogen Idec within the last 3 years. There was no funding-body involvement in the writing or submission of this article.

Acknowledgements I am very grateful to Hugh Bostock FRS for his helpful criticism of a version of the manuscript, and also for providing a figure on the use of threshold-electrotonus.

References Alvin ˜a K, Khodakhah K. The therapeutic mode of action of 4aminopyridine in cerebellar ataxia. Journal of Neuroscience 2010;30:7258–68.

Potential therapeutic mechanism of K + channel block for MS Arroyo EJ, Xu T, Grinspan J, et al. Genetic dysmyelination alters the molecular architecture of the nodal region. Journal of Neuroscience 2002;22:1726–37. Asselman P, Chadwick DW, Marsden DC. Visual evoked responses in the diagnosis and management of patients suspected of multiple sclerosis. Brain 1975;98:261–82. Baker MD. Axonal flip-flops and oscillators. Trends in Neuroscience 2000;23:514–9. Baker MD. Ion currents and axonal oscillators: a possible biophysical basis for positive signs and symptoms in multiple sclerosis. In: Waxman SG, editor. Multiple sclerosis as a neuronal disease. San Diego, USA: Elsevier Academic Press; 2005. p. 85-100. Baker M, Bostock H. Ectopic activity in demyelinated spinal root axons of the rat. Journal of Physiology 1992;451:539–52. Baker M, Bostock H, Grafe P, Martius P. Function and distribution of three types of rectifying channel in rat spinal root myelinated axons. Journal of Physiology 1987;383:45–67. Baker MD, Chen YC, Shah SU, Okuse K. In vitro and intrathecal siRNA mediated KV1.1 knock-down in primary sensory neurons. Molecular and Cellular Neuroscience 2011;48:258–65. Barrett EF, Barrett JN. Intracellular recording from vertebrate myelinated axons: mechanism of the depolarizing afterpotential. Journal of Physiology 1982;323:117–44. Bostock H, Baker M. Evidence for two types of potassium channel in human motor axons in vivo. Brain Research 1988;462:354–8. Bostock H, Grafe P. Activity-dependent excitability changes in normal and demyelinated rat spinal root axons. Journal of Physiology 1985;365:239–57. Bostock H, Sears TA. The internodal axon membrane: electrical excitability and continuous conduction in segmental demyelination. Journal of Physiology 1978;280:273–301. Bostock H, Sears TA, Sherratt RM. The effects of 4-aminopyridine and tetraethylammonium ions on normal and demyelinated mammalian nerve fibres. Journal of Physiology 1981;313:301–15. Bostock H, Sherratt RM, Sears TA. Overcoming conduction failure in demyelinated nerve fibres by prolonging action potentials. Nature 1978;274:385–7. Brown DA, Passmore GM. Neural KCNQ (KV7) channels. British Journal of Pharmacology 2009;156:1185–95. Calvin WF, Devor M, Howe JF. Can neuralgias arise from minor demyelination? Spontaneous firing, mechanosensitivity, and afterdischarge from conducting axons Experimental Neurology 1982;75:755–63. Chiu SY, Ritchie JM, Rogart RB, Stagg D. A quantitative description of membrane currents in rabbit myelinated nerve. Journal of Physiology 1979;292:149–66. Corrette BJ, Repp H, Dreyer F, Schwarz JR. Two types of fast K + channels in rat myelinated nerve fibres and their sensitivity to dendrotoxin. Pflugers Archiv—European Journal of Physiology 1991;418:408–16. Davis FA, Stefoski D, Rush J. Orally administered 4-aminopyridine improves clinical signs in multiple sclerosis. Annals of Neurology 1990;27:186–92. Dedek K, Kunath B, Kananura C, et al. Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K + channel. Proceedings of the National Academy of Sciences USA 2001;98:12272–7. Devaux J, Alcaraz G, Grinspan J, et al. Kv3.1b is a novel component of CNS nodes. Journal of Neuroscience 2003;23:4509–18. Devaux J, Gola M, Jacquet G, Crest M. Effects of K + channel blockers on developing rat myelinated CNS axons: identification of four types of K + channels. Journal of Neurophysiology 2002;87:1376–85. Devaux JJ, Kleopa KA, Cooper EC, Scherer SS. KCNQ2 is a nodal K + channel. Journal of Neuroscience 2004;24:1236–44. Dolly JO, Parcej DN. Molecular properties of voltage-gated K + channels. Journal of Bioenergetics and Biomembranes 1996;28: 231–253.

279 Dubois JM. Evidence for the existence of three types of potassium channels in the frog Ranvier node membrane. Journal of Physiology 1981;318:297–316. Eng DL, Gordon TR, Kocsis JD, Waxman SG. Development of 4-AP and TEA sensitivities in mammalian myelinated nerve fibres. Journal of Neurophysiology 1988;60:2168–79. Gordon TR, Kocsis JD, Waxman SG. Evidence for the presence of two types of potassium channels in the rat optic nerve. Brain Research 1988;447:1–9. Hadavi S, Baker MD, Dobson R. Fampridine in multiple sclerosis. Multiple Sclerosis and Related Disorders, submitted for publication [MSARD-D-12-00107, submitted 2 November 2012]. Halliday AM, McDonald WI, Mushin J. Delayed pattern-evoked responses in optic neuritis in relation to visual acuity. Transactions of the Ophthalmological Societies of the United Kingdom 1973;93:315–24. Harvey AL. Twenty years of dendrotoxins. Toxicon 2001;39:15–26. Hildebrand C, Mohseni S. The structure of myelinated axons in the CNS. In: Waxman SG, editor. Multiple sclerosis as a neuronal disease. San Diego, USA: Elsevier Academic Press; 2005. p. 1-28. Howe JR, Ritchie JM. On the active form of 4-aminopyridine: block of K + currents in rabbit Schwann cells. Journal of Physiology 1991;433:183–205. Jack JJB, Redman SJ, Wong K. Modifications to synaptic transmission at group Ia synapses on cat spinal motoneurones by 4aminopyridine. Journal of Physiology 1981;321:111–26. Johnston J, Forsythe ID, Kopp-Scheinpflug C. Going native: voltagegated potassium channels controlling neuronal excitability. Journal of Physiology 2010;588:3187–200. Koh DS, Jonas P, Vogel W. Na + -activated K + channels localized in the nodal region of myelinated axons of Xenopus. Journal of Physiology 1994;479:183–97. Mourre C, Chernova MN, Martin-Eauclaire MF, et al. Distribution in rat brain of binding sites of kaliotoxin, a blocker of KV1.1 and KV1.3 a-subunits. Journal of Pharmacology and Experimental Therapeutics 1999;291:943–52. Nordin M, Nystr¨ om B, Wallin U, Hagbarth KE. Ectopic sensory discharges and paresthesiae in patients with disorders of peripheral nerves, dorsal roots and dorsal columns. Pain 1984;20:231–45. Perge JA, Koch K, Miller R, et al. How the optic nerve allocates space, energy capacity, and information. Journal of Neuroscience 2009;29:7917–28. Poliak S, Peles E. The local differentiation of myelinated axons at nodes of Ranvier. Nature Reviews Neuroscience 2003;4:968–80. Rasband MN, Trimmer JS, Peles E, et al. K + channel distribution and clustering in developing and hypomyelinated axons of the optic nerve. Journal of Neurocytology 1999;28:319–31. Rasband MN, Shrager P. Ion channel sequestration in central nervous system axons. Journal of Physiology 2000;525:63–73. Rasband MN, Trimmer JS. Developmental clustering of ion channels at and near the node of Ranvier. Developmental Biology 2001;236:5–16. Rasband MN, Trimmer JS, Schwarz TL, et al. Potassium channel distribution, clustering, and function in remyelinating rat axons. Journal of Neuroscience 1998;18:36–47. Reid G, Scholz A, Bostock H, Vogel W. Human axons contain at least five types of voltage-dependent potassium channel. Journal of Physiology 1999;518:681–96. R¨ oper J, Schwarz JR. Heterogeneous distribution of fast and slow potassium channels in myelinated rat nerve fibres. Journal of Physiology 1989;416:93–110. Rossini PM, Pasqualetti P, Pozzilli C, et al. Fatigue in progressive multiple sclerosis: results of a randomized, double-blind, placebo-controlled, crossover trial of oral 4-aminopyridine. Multiple Sclerosis 2001;7:354–8. Rushton WA. A theory of the effects of fibre size in medullated nerve. Journal of Physiology 1951;115:101–22.

280 Safronov BV, Kampe K, Vogel W. Single voltage-dependent potassium channels in rat peripheral nerve membrane. Journal of Physiology 1993;460:675–91. Scherokman BJ, Selhorst JB, Waybright EA, et al. Improved optic nerve conduction with ingestion of ice water. Annals of Neurology 1985;17:418–9. Schwarz JR, Glassmeier G, Cooper EC, et al. KCNQ channels mediate IKs, a slow K + current regulating excitability in the rat node of Ranvier. Journal of Physiology 2006;573:17–34. Shah MM, Migliore M, Valencia I, et al. Functional significance of axonal KV7 channels in hippocampal pyramidal neurons. Proceedings of the National Academy of Sciences USA 2008;105:7869–74. Sherratt RM, Bostock H, Sears TA. Effects of 4-aminopyridine on normal and demyelinated mammalian nerve fibres. Nature 1980;283:570–2. Shi R, Blight AR. The differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord. Neuroscience 1997;77:553–62. Shi R, Kelly TM, Blight AR. Conduction block in acute and chronic spinal cord injury: different dose response characteristics for reversal by 4-aminopyridine. Expimental Neurology 1997;148: 495–501. Smart SL, Lopantsev V, Zhang CL, et al. Deletion of the KV1.1 potassium channel causes epilepsy in mice. Neuron 1998;20: 809–819. Smith KJ, Felts PA, John GR. Effects of 4-aminopyridine on demyelinated axons, synapses and muscle tension. Brain 2000;123:171–84. Smith KJ, Waxman SG. The conduction properties of demyelinated and remyelinated axons. In: Waxman SG, editor. Multiple sclerosis as a neuronal disease. San Diego, USA: Elsevier Academic Press; 2005. p. 85-100. Smith KJ, Waxman SG. The conduction properties of demyelinated and remyelinated axons. In: Waxman SG, editor. Multiple

M.D. Baker sclerosis as a neuronal disease. Elsevier Academic Press; 2005. p. 85–100. Sospedra M, Martin R. Immunology of multiple sclerosis. Annual Review of Immunology 2005;23:683–747. Thesleff S. Aminopyridines and synaptic transmission. Neuroscience 1980;5:1413–9. Tomlinson SE, Bostock H, Grinton B, et al. In vivo loss of slow potassium channel activity in individuals with benign familial neonatal epilepsy in remission. Brain 2012;135:3144–52. Tomlinson SE, Tan SV, Kullmann DM, et al. Nerve excitability studies characterize KV1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1. Brain 2010;133:3530–40. Ulzheimer JC, Peles E, Levinson SR, Martini R. Altered expression of ion channel isoforms at the node of Ranvier in P0-deficient myelin mutants. Molecular and Cellular Neuroscience 2004;25:83–94. Wang H, Kunkel DD, Martin TM, et al. Heteromultimeric K + channels in terminal and juxtaparanodal regions of neurons. Nature 1993;365:75–9. Wang H, Kunkel DD, Schwartzkroin PA, Tempel BL. Localization of KV1.1 and KV1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. Journal of Neuroscience 1994;14:4588–99. Wang H-S, Pan Z, Shi W, et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. Science 1998;282:1890–3. Wu Z-Z, Li D-P, Chen S-R, Pan H-L. Aminopyridines potentiate synaptic and neuromuscular transmission by targeting the voltage-activated calcium channel b subunit. Journal of Biological Chemistry 2009;284:36453–61. Zuberi SM, Eunson LH, Spauschus A, et al. A novel mutation in the human voltage-gated potassium channel gene (KV1.1) associates with episodic ataxia type 1 and sometimes with partial epilepsy. Brain 1999;122:817–25.