A Way out for Neuropathic Pain?

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REVIEW ARTICLE published: 04 August 2009 MOLECULAR NEUROSCIENCE doi: 10.3389/neuro.02.010.2009 Enhancing M currents: a way out for neuropathic pain?

Ivan Rivera-Arconada, Carolina Roza and Jose A. Lopez-Garcia*

Departamento de Fisiología, Ediﬁ cio de Medicina, Universidad de Alcala, Madrid, Spain

Edited by: Almost three decades ago, the M current was identifi ed and characterized in frog sympathetic Bernard Attali, Tel Aviv University, Israel neurons (Brown and Adams, 1980). The years following this discovery have seen a huge Reviewed by: progress in the understanding of the function and the pharmacology of this current as well as Holger Lerche, Universitätsklinikum Ulm, Germany on the structure of the underlying ion channels. Therapies for a number of syndromes involving Dirk Isbrandt, Zentrum für Molekulare abnormal levels of excitability in neurons are benefi ting from research on M currents. At present, Neurobiologie Hamburg, Germany the potential of M current openers as analgesics for neuropathic pain is under discussion. Here *Correspondence: we offer a critical view of existing data on the involvement of M currents in pain processing. We Jose A. Lopez-Garcia, Departamento believe that enhancement of M currents at the site of injury may become a powerful strategy de Fisiología, Edifi cio de Medicina, Universidad de Alcala, Alcala de to alleviate pain in some peripheral neuropathies. Henares, 28871 Madrid, Spain. Keywords: potassium current, KCNQ, Kv7, pain, analgesia, pain therapy, neuropathy e-mail: [email protected]

INTRODUCTION systems and to evaluate their potential as targets for analgesia under Potassium channels constitute a very wide family of ionic channels conditions of neuropathy. that are present in most biological entities. Individual subunits can combine to form heteromeric channels and post-transcriptional M CURRENTS: FUNCTION AND MODULATION modifi cations of these subunits are possible yielding a great variety In 1998, the molecular substrates for the M current were fi rst of potassium currents. Genetic defects in genes coding potassium identifi ed as KCNQ2 and KCNQ3 channels (Wang et al., 1998) channel subunits have been related to human diseases indicating the and later on, KCNQ4 and KCNQ5 were also found to contribute great importance that they have in cellular and systems physiology to generate M currents (Kubisch et al., 1999; Lerche et al., 2000; (Kullmann, 2002). Schroeder et al., 2000). The remaining member of the family, In excitable cells potassium channels are responsible for the KCNQ1, is heavily expressed in the heart but not in nervous sys- control and regulation of excitability. The activity of individual tem (Jentsch, 2000; Robbins, 2001). The present nomenclature for potassium channels or combinations of them can determine rest- ion channels has renamed them as Kv7.1, Kv7.2, Kv7.3, Kv7.4 and ing potential, membrane resistance, action potential shape and fi r- Kv7.5 respectively and we will use this nomenclature from here ing adaptation. In addition, the antinociceptive action of several onwards (Gutman et al., 2003). endogenous mediators like opioids as well as some anesthetics, are Functional Kv7 channels require homomeric or heteromeric partly mediated by actions on potassium channels (Ocana et al., assemblies of four subunits. However, only the Kv7.3 subunit is 2004). For these reasons, there is an increasing interest in study- able to form heteromeric channels with other members of the ing the involvement of potassium channels in the sensitization of family (for review see Robbins, 2001). Kv7 proteins consist of six nociceptive systems (Hu et al., 2006). transmembrane domains and their structure has been studied in

M currents (IM) and the underlying KCNQ or Kv7 channels have detail (Jentsch, 2000). Several sites of functional relevance have been been related to several channelopathies that involve hyperexcitabil- described including phosphorylation sites (Schroeder et al., 1998; ity in neurons of various areas of the nervous system. These include Surti et al., 2005), a calmodulin-binding site (Etxeberria et al., 2008; benign neonatal epilepsy and other forms of epilepsy, myokymia Yus-Najera et al., 2002) and binding sites for AKAP and ankyrin- and peripheral nerve excitability (Maljevic et al., 2008). Several cues G (Hoshi et al., 2003; Pan et al., 2006). The general functions of suggest that opening of M currents with specifi c anticonvulsants these channels in the nervous system have been recently reviewed may be a valid target for analgesia in neuropathic pain states. In elsewhere (Brown and Passmore, 2009). fi rst place, anticonvulsants with different mechanisms of action like pregabalin and gabapentin are being successfully used for neu- M CURRENT AND NEURONAL EXCITABILITY ropathic pain therapy. In second place, basic research is providing As expected from the various combinations of proteins that can evidence showing abundant expression of functional M currents form functional currents, there is not a single M current but in primary afferents and the spinal cord, both key elements for rather a family of them that share some major traits. The classi- nociceptive processing. Finally, some M current modulators are cal IM is a voltage dependent non-inactivating potassium current showing promising signs of pain relieve in animal models of neu- with a slow activation process which starts at membrane poten- ropathic pain. tials close to rest (Brown and Yu, 2000). Molecular variations Here we review experimental work that has contributed to defi ne affect channel conductance and the sensitivity to modulators M currents, to assess their presence and function in nociceptive (Robbins, 2001).

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Because of its low activation threshold, it can contribute to Bradykinin has been shown to increase the excitability of SCG maintain the resting potential together with other conductances neurons inhibiting Kv7. This effect of bradykinin is mediated by (Lamas et al., 2002). At resting membrane potential there are very B2 receptors and the activation of G alpha q/11 proteins acting via few operative ionic currents and therefore a large membrane resist- phospholipase C (Cruzblanca et al., 1998; Jones et al., 1995). This, ance. In these conditions, although Kv7 channels are only starting in turn may cause increases in calcium concentration and closure to open, the small currents generated may have a large impact on of Kv7 channels (Brown et al., 2007; Delmas et al., 2002). resting potential. In agreement with this theoretical interpreta- Several other endogenous mediators like serotonin and nerve tion, modulation of Kv7 channels has been reported to produce growth factor have been implicated in IM reduction (Jia et al., 2008; changes in resting membrane potentials and/or input resistance McCormick and Williamson, 1989). Similarly, activation of CB1 in many neuronal types (Brown and Adams, 1980; Koyama and cannabinoid receptors (Schweitzer, 2000), P2Y purinergic recep- Appel, 2006; Lawrence et al., 2006; Lechner et al., 2003; Murase tors (Adams et al., 1982; Filippov et al., 2006; Zaika et al., 2007) et al., 1986; Nowak and Macdonald, 1982, 1983a,b; Otto et al., and metabotropic glutamate receptors (Charpak et al., 1990) may 2002; Passmore et al., 2003; Rivera-Arconada and Lopez-Garcia, reduce M currents.

2005; Romero et al., 2004; Wladyka and Kunze, 2006; Yue and Yaari, In contrast to these excitatory effects mediated by IM depression, 2004). As a consequence of the effects on resting potential, Kv7 opioids and somatostatin have been reported to reduce neuronal channels (particularly those containing Kv7.2 subunits) have been excitability by potentiating the current. Activation of kappa recep- implicated in the modulation of neurotransmitter release (Lechner tors by modulators like dynorphin and nociceptin has been shown et al., 2003; Luisi et al., 2009; Martire et al., 2004, 2007). to depress the excitability of CA1 neurons (Madamba et al., 1999a,b; M currents are active at potentials below the threshold for action Moore et al., 1994). In this region of the CNS, somatostatin (and potential initiation and tend to increase rapidly in magnitude with corticostatin) has been shown to augment the M current (de Lecea depolarization. This creates a force opposing to depolarization, et al., 1996; Moore et al., 1988) activating the subtype 4 receptor delaying or precluding the fi ring of action potentials due to synaptic (Qiu et al., 2008) and a subsequent pathway implicating arachidonic activation. Because the current does not inactivate but remains acid metabolites (Schweitzer et al., 1990). In neurons of the soli- active during prolonged periods of depolarization it tends to reduce tary tract somatostatin produces hyperpolarization and depresses fi ring frequency. Modulation of Kv7 channels has an important excitability increasing Kv7 currents (Jacquin et al., 1988). impact on neuronal excitability as reported for many native neu- With the exception of muscarine and substance P, there is no rons. Block of Kv7 channels leads to a decrease in rheobase and a evidence that the other mentioned receptors and endogenous reduction in the spike frequency adaptation reducing the interspike modulators may act on M currents to regulate pain processing. interval, while channel opening facilitation tends to produce oppo- Of special interest in our context is the recent work performed on site effects (Filippov et al., 2006; Hetka et al., 1999; Koyama and small diameter dorsal root ganglion cells (presumed nociceptors) Appel, 2006; Lawrence et al., 2006; Lechner et al., 2003; Nowak and showing that activation of Mas-related gene receptor D and pro- Macdonald, 1983a; Otto et al., 2002; Passmore et al., 2003; Peretz tease-activated receptor 2 cause inhibition of M currents and an et al., 2005; Rivera-Arconada and Lopez-Garcia, 2005; Wladyka abnormal excitability in nociceptive pathways which may be related and Kunze, 2006; Yue and Yaari, 2004), but not always (Miles et al., to the origin of naturally occurring hyperalgesia after infl ammation 2005; Romero et al., 2004). (Crozier et al., 2007; Linley et al., 2008).

ENDOGENOUS MODULATION OF M CURRENTS EXOGENOUS MODULATORS OF M CURRENTS Kv7 channels are modulated by a variety of endogenous compounds Selective agonists and antagonists are extremely useful tools to like neurotransmitters and peptides via the activation of intracel- evaluate the roles of ion channels and receptors at the systems level. lular cascades of second messengers. Interestingly, most modulators The ﬁ rst compounds known to act as M current modulators with produce a reduction of IM and hence tend to increase the excitability a certain degree of potency and selectivity were linopirdine (chan- of the neurons affected (Brown and Yu, 2000). nel blocker) and retigabine (channel opener). Although Kv7 chan- Activation of muscarinic acetylcholine receptors was the ﬁ rst nels are sensitive to tetraethylammonium (Hadley et al., 2000) this pathway reported for M current modulation and the name of compound is not selective and blocks a variety of other potassium the current is related to this fact (Brown and Adams, 1980). conductances. Linopirdine probably exerts its effect on Kv7 chan-

Cholinergic inhibition of IM is a widespread mechanism and it nels at the external side of the membrane (Costa and Brown, 1997). has been conﬁ rmed in many areas of the nervous system includ- Important residues for the effect of retigabine on Kv7 channels are ing human cortical neurons (McCormick and Williamson, 1989). located within the pore region (Lange et al., 2009; Schenzer et al., Cholinergic suppression of the current seems to be mediated by 2005; Wuttke et al., 2005). M1 receptors in most neuronal types tested (Marrion et al., 1989; Linopirdine [3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2- Romero et al., 2004; Selyanko et al., 2000; Shen et al., 2005) but one, DUP996] was described as a cognition enhancer that increases not in all of them (Rouse et al., 2000). The effect of muscarinic stimulus evoked neurotransmitter release in hippocampus without receptors on IM seems to be mediated by G-proteins of the Gq or altering basal release. For this reason, it was proposed for the treat- G11 class (Caulﬁ eld et al., 1994). In addition, it has been shown ment of Alzheimer’s disease (Tam and Zaczek, 1995). The mecha- that PIP2 is a necessary cofactor for Kv7 function and that activa- nism of action of linopirdine is mostly mediated by Kv7 channel tion of muscarinic receptors produces PIP2 depletion (Suh and closure (Aiken et al., 1995), although effects on other potassium Hille, 2005). channels and receptors cannot be excluded (Lamas et al., 1997;

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Schnee and Brown, 1998). Some analogs of linopirdine show similar cyclooxygenase activity may lead to obtain potent and subunit properties (Zaczek et al., 1998). Among them XE991 [10,10-bis(4- specifi c Kv7 modulators that could be applicable to pain treat- pyridinylmethyl)-9(10H)-anthracenone] has been widely used for ment (Peretz et al., 2007). Kv7 channel studies because it has greater affi nity and selectivity values than linopirdine for native M currents (Robbins, 2001; Kv7 CHANNELS AND M CURRENTS IN SENSORY AFFERENTS Schroder et al., 2001; Wang et al., 1998). These compounds also Primary afferents are highly specialized elements of sensory systems block cardiac Kv7.1 channels at low concentrations, however in comprising four differentiated domains with specialized functions, native cardiac cells Kv7.1 associates with other accessory subunits i.e., peripheral nerve endings, conducting axons, central terminals that render the resulting current less sensitive to XE991 (Wang and cell bodies. Ion channels are distributed in an orderly and et al., 2000). inhomogeneous form in each domain contributing in a decisive Kv7 channel blockers have been widely used for the identifi cation way to the signaling properties of individual fi bers. Alterations in of M currents in several neuronal types. In many neurons includ- the quantitative expression or the orderly distribution of ion chan- ing dorsal root ganglion and spinal cord neurons, application of nels induced by injury may have profound effects in these signal- XE991 produces depolarization and an increase in the fi ring fre- ing properties. The paragraphs bellow describe the experimental quency to depolarizing stimuli (Aiken et al., 1995; Alaburda et al., evidence supporting the presence of Kv7 channels in afferents and 2002; Lawrence et al., 2006; Otto et al., 2002, 2006; Passmore et al., the functional consequences that may derive. 2003; Peters et al., 2005; Rivera-Arconada and Lopez-Garcia, 2005; Wladyka and Kunze, 2006; Yue and Yaari, 2004). However some PERIPHERAL NERVE ENDINGS neurons show weak responses to XE991 even if they have Kv7 chan- Peripheral terminals contain the machinery required for trans- nels (Miles et al., 2005; Rivera-Arconada and Lopez-Garcia, 2005; duction and coding of external stimuli. Several potassium con- Romero et al., 2004). This may be due to the subunit composition ductances contribute to shape receptor potentials and to modulate of the different channels or to the expression of other dominant coding properties of individual afferents (Belmonte and Viana, currents in the same neuron. In many studies, Kv7 channel blockers 2008). Several lines of evidence suggest the presence of Kv7 chan- have been used as antagonist to test the specifi city of retigabine on nels or the existence of M currents at specialized receptor endings M currents (Blackburn-Munro and Jensen, 2003; Dost et al., 2004; (Barnes, 1994; Buniel et al., 2008). Wladyka et al. (2008) studied the Passmore et al., 2003; Rivera-Arconada and Lopez-Garcia, 2005, sensory terminals of arterial baroreceptors and found expression 2006; Rivera-Arconada et al., 2004; Roza and Lopez-Garcia, 2008; of Kv7.2/3/5 channel subunits as well as modulation of pressure– Yue and Yaari, 2004). response relations by retigabine and XE991. These results indicate Retigabine [D23129, ethyl N-(2-amino-4-(4-fl uorobenzylamino)- that Kv7 channels are likely contributors to coding process, at least phenyl] activates M currents produced by heterologous expression of in certain receptors. Kv7.2/3 (Main et al., 2000; Rundfeldt and Netzer, 2000b; Wickenden Cutaneous sensitivity has been explored using the skin-nerve et al., 2000). At concentrations between 0.1 and 10-µM retigab- preparation by several groups. We found that only a small propor- ine modifi es the voltage dependence of these channels, increases tion of Aδ and C afferent fi bers responding to mechanical or chemi- the activation rate, slows its inactivation and favors the open channel cal stimuli were responsive to XE991 and none of them responded confi guration (Tatulian and Brown, 2003). Interestingly retigabine to retigabine (Roza and Lopez-Garcia, 2008). We concluded that does not affect currents mediated by Kv7.1 channels. At larger con- even if present in intact terminals, M currents must have a minor centrations, retigabine has been found to interact with GABA-ergic role at coding of acute nociceptive stimuli of cutaneous origin. In systems in some studies (Kapetanovic et al., 1995; Otto et al., 2002; agreement with this observation, cutaneous application of XE991 Rundfeldt and Netzer, 2000a; van Rijn and Willems-van Bree, 2003) to live mice did not produce mechanical or thermal hyperalge- but not in others (Hetka et al., 1999). sia (Linley et al., 2008). However, preliminary data from another Application of retigabine produces hyperpolarization and pro- group using the skin-nerve preparation (Passmore and Brown, motes spike frequency adaptation in several neuronal types includ- 2007) reported a depressant action of retigabine on noxious heat ing spinal cord neurons (Rivera-Arconada and Lopez-Garcia, 2005), and mechanical responses. It is possible that differences between dorsal root ganglion (Passmore et al., 2003), cortical (Hetka et al., these two data sets were due to the use of different animals (mice 1999; Otto et al., 2002) and hippocampal neurons (Yue and Yaari, and rats respectively) or different types of sensory neurons, but 2004). Retigabine also produces a reduction in neurotransmitter further clarifi cation will be required. release induced by depolarization in synaptosomes (Luisi et al., 2009; Martire et al., 2004, 2007). CONDUCTING AXONS Due to their interesting and promising pharmacological prop- Conduction of action potentials through myelinated and unmyeli- erties, new Kv7 channel openers have been developed recently nated axons involves two separated sets of mechanisms. At present, and Kv7 channel opener properties have been reported in exist- no data exist on the distribution and function of Kv7 channels along ing compounds. Among these are fl upirtine, BMS-204352, unmyelinated axons although electrophysiological experiments diclofenac, ICA-27243, several acrylamides and Zinc pyrithione suggest the expression of functional M currents in this fi ber type. (Dalby-Brown et al., 2006, Xiong et al., 2008). Diclofenac and In myelinated fi bers, nodes of Ranvier at periodical interruptions meclofenamic acid are commonly used anti-infl ammatory drugs of the myelin sheath enable saltatory conduction. Nodes are highly which activate Kv7 channels (Peretz et al., 2005). The design of specialized structures enriched in sodium and potassium channels new derivates that maintain Kv7 enhancing properties without (Poliak and Peles, 2003). It has been shown that Kv7.2 channels are

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expressed in nodes of peripheral axons colocalized with sodium 2008; Passmore et al., 2003). Functional M currents with standard channels where they share an ankyrin-G-based mechanism of bi ophysics and pharmacological characteristics were found in retention (Devaux et al., 2004; Pan et al., 2006). The Kv7.3 subu- large and small cells, some of them sensitive to capsaicin and nit has also been detected in paranodes and Schmidt–Lanterman therefore potential nociceptive fi bers. Immunofl uorescent analy- incisures in a relative small percentage of fi bers. It is noteworthy sis showed that Kv7.2/3/5 subunits were present in ganglion cells that Kv7.2 and Kv7.3 do not colocalize even when they are found probably producing functional channels of all possible combina- in the same fi bers. Therefore, it is likely that Kv7.2 homomers con- tions. Modulation of M currents in dorsal root ganglion cells has stitute the major contribution of Kv7 channels to nodal potassium been suggested as a mechanism to induce hyperexcitability during currents, at least in peripheral fi bers. infl ammation (Linley et al., 2008). A variety of nodal and perinodal potassium channels may contribute to limit spike duration and frequency adaptation. Recently, M CURRENTS, AFFERENT FIBERS AND NEUROPATHY Kv7.2 channels have been proposed to mediate the IKs (Schwarz Afferent fi bers are known to play a fundamental role in the origin et al., 2006), a slow potassium current previously detected at nodes and maintenance of neuropathic states. After injury, a percentage (Dubois, 1981). XE991 has been shown to block IKs in single sci- of myelinated and unmyelinated afferents may degenerate and die. atic myelinated axons from rats maintained in vitro, the functional The surviving afferents may regenerate and innervate new targets consequences being a decreased current threshold and a reduced or fail to regenerate and form a neuroma (Lisney, 1989). In the adaptation to intracellular current pulses. Schwarz et al. (2006) area of damage, fi bers lose their myelin sheath and express a vari- showed that under in vivo conditions XE991 increase the excitability ety of membrane proteins such as transductors, receptors and ion of motor axons. Accordingly, myokymia (neuromyotonia) has been channels normally not present in axons (Zimmermann, 2001). As described in connection with mutations affecting gating of Kv7.2 a result these areas acquire the capacity to generate ectopic action (Dedek et al., 2001; Wuttke et al., 2007). It is not known how this potentials either spontaneously or in response to stimuli of different may relate to sensory conduction under physiological conditions modalities. Similarly, axotomized dorsal root ganglion cells develop although no sensory defi cits have been associated to mutations in hyperexcitability and spontaneous activity (Amir et al., 2005). genes coding Kv7 channels. Sodium channels are key elements for electrogenesis and a large number of observations indicate that they play an essential role CENTRAL TERMINALS OF PRIMARY AFFERENTS in the development of ectopic activity (Devor, 2006). A role for Central terminals are specialized in the release of neurotransmitters potassium channels in ectopia has been anticipated on the basis of onto second order dorsal horn neurons. Neurotransmitter release large increases in spontaneous activity produced by classic potas- can be dynamically controlled by presynaptic contacts and local sium channel blockers such as 4-aminopyridine and tetraethylam- membrane potential. In addition, it has been proposed that exces- monium (Devor, 1983). Recent work indicates that Kv7 channels sive depolarization at central terminals may induce backfi ring in may infl uence hyperexcitability of neuromatose fi bers. We used the afferents contributing to peripheral sensitization (Willis, 1999). saphenous skin-nerve and nerve-end neuroma preparations to Therefore, the control of membrane potential and excitability at compare the effects of retigabine and XE991 on the coding prop- this point is crucial for sensory transmission. erties of intact and neuromatose nerve endings (Roza and Lopez- Indirect electrophysiological observations on populations of Garcia, 2008). To our surprise, retigabine was devoid of effect on afferent fi bers indicate that functional Kv7/M currents are present responses of intact Aδ and C-fi bers to the stimulation of their recep- in central terminals as well (Rivera-Arconada and Lopez-Garcia, tive fi eld. However responses of neuromatose Aδ and C-fi bers where 2006). Retigabine produces hyperpolarization of terminals and completely blunted by retigabine. In addition, XE991 blocked the reduces the excitability of a range of afferent fi bers including myeli- inhibitory effect of retigabine. Mechanistic studies indicated that nated and unmyelinated afferents. XE991 has no effect on the ter- retigabine hyperpolarized the areas of ectopic discharge generation minals when applied alone but blocks the effects of retigabine. decreasing the probability of fi ring action potentials. This mechanism could operate to suppress activity independently of the nature DORSAL ROOT GANGLION CELLS of the stimulus and fi ber type. These fi ndings suggest that Kv7 chan- The cell bodies of primary afferents are grouped in dorsal root nels gain an extraordinary important role in the control of ectopic ganglia. The function of this compartment of primary afferents is discharges which may result from a complex rearrangement of the not as clear as for the other components which are directly involved ion channels expressed at ectopic areas and/or an altered expression in the transmission of information from the periphery to the spinal of Kv7 subunits as suggested by preliminary work (Wickenden et al., cord. Action potential invasion of the soma is not required for trans- 2002). A similar conclusion may be reached from work on isolated mission along the main process and there are no synapses in dorsal fi bers from human beings suffering from peripheral vascular dis- root ganglion. Somas of primary afferents are classically viewed as ease or polyneuropathy (Lang et al., 2008). This altered sensitivity metabolic factories dedicated to supporting the conducting axon. to retigabine in neuromatose fi bers generates a peripheral target However, the cell soma is normally invaded by action potentials of interest for analgesia in some forms of neuropathy. running in the main process and this may represent some functional advantages (Amir and Devor, 2003). Kv7 CHANNELS AND M CURRENTS IN THE SPINAL CORD Among a variety of sodium and potassium ion channels which The spinal cord receives sensory inputs from the periphery becom- confer the membrane full active electrical properties, the somata ing the fi rst relay center for nociceptive information. The spinal of dorsal root ganglion cells express Kv7 channels (Linley et al., cord processes the original sensory messages integrating descending

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signals from the brain in order to produce two main forms of that retigabine produces hyperpolarization of dorsal horn neurons output: (1) sensory output to inform brain centers which eventually and a decrease in their intrinsic excitability which manifests as will generate the sensation of pain and (2) motor output to organize an augmented rheobase and an increased spike frequency adapta- refl ex activity. Whereas sensory outputs are generated in the dorsal tion. XE991 produced a complete reversion of retigabine effects horn, the motor output necessarily requires further processing in indicating a specifi c action on Kv7 channels; however, XE991 had the ventral horn (Willis and Coggeshall, 2004). In this strategic a very weak excitatory effect when applied on its own (Rivera- position, the spinal cord is the structure most frequently studied Arconada and Lopez-Garcia, 2005). Interestingly, all studies show to understand central mechanisms of pain processing. a large proportion of dorsal horn neurons responding to retigabine Recently several studies have examined the expression of Kv7 suggesting that Kv7 channels are commonly expressed by dorsal subunits in the spinal cord although unfortunately a systematic horn neurons. description of the laminar distribution is not available yet. Despite the fi rst failed attempts to detect the presence of Kv7 channels M CURRENTS IN MOTONEURONS (Biervert et al., 1998; Smith et al., 2001; Yang et al., 1998), Dedek The presence of Kv7 at axon initial segments of motoneurons et al. (2001) demonstrated the presence of Kv7.2 and Kv7.3 subunits (Devaux et al., 2004; Pan et al., 2006), a structure of great impor- in both the dorsal and ventral horn of the mouse spinal cord using tance in the generation and the transmission of action potentials, in situ hybridization which was later confi rmed by immunoblots strongly argue in favor of a decisive role of these channels at regu- and immunohistochemical techniques (Devaux et al., 2004). These lating the motor output of the spinal cord. Electrophysiological authors showed the presence of Kv7.2 channels in axon initial seg- studies using M current modulators also indicate the presence of ments throughout the gray matter. The Kv7.3 subunit was also functional M currents in spinal motoneurons regulating its intrin- found in the gray matter of the spinal cord associated to axon sic excitability. In turtle motoneurons both muscarine and XE991 initial segments and nodes of motoneurons (Pan et al., 2006). The increased the fi ring frequency of action potentials by reducing an presence of Kv7 subunits in axon initial segments confers a great M-like current (Alaburda et al., 2002). Retigabine has been shown relevance to Kv7 channels in the neuronal integration of synaptic to depress nociceptive spinal refl exes having only minor effects inputs since this region of the axon constitutes a subcellular loca- on non-nociceptive refl exes (Rivera-Arconada et al., 2004). As in tion of great importance in the generation of action potentials the dorsal horn, retigabine reduces responses to the activation of (Stuart et al., 1997). In addition, Kv7.3 channels may be expressed nociceptive afferents and wind-up. Motoneurons show a similar by astrocytes (Devaux et al., 2004). response to retigabine as that reported for dorsal horn neurons, i.e., Unpublished observations from our laboratory indicate that a decrease in excitability. However, XE991 produced extraordinary Kv7.5 subunits are also abundantly expressed in spinal cord neu- large increases in excitability compared to the effects seen in dorsal rons. We have used two different antisera previously used for immu- horn neurons. This could be due to a differential expression of Kv7 nohistochemical detection of Kv7.5 channels in nervous tissue subunits in both neuronal types. Here again the effects of retigabine (Caminos et al., 2007; Yus-Najera et al., 2003) and obtained the were prevented by XE991 coapplication indicating a selective action same picture: clear staining of a considerable proportion of dorsal of the modulators on Kv7 channels. All motoneurons respond read- horn (deep and superfi cial) and ventral horn neurons (inter- and ily to the modulators, indicating a widespread expression of the motorneurons) as well as diffuse staining restricted to superfi cial channels (Rivera-Arconada and Lopez-Garcia, 2005). dorsal areas that could label the central terminals of afferent fi bers. The existence of Kv7.5 proteins in spinal cord neurons favors the M CURRENTS, THE SPINAL CORD AND NEUROPATHY possibility of producing heteromeric channels and adding func- There is abundant evidence supporting the concept of central sen- tional and pharmacological diversity to spinal M currents. sitization (Ji et al., 2003). Although spinal neurons (and neurons in other brain centers related to pain processing) are not directly M CURRENTS IN DORSAL HORN NEURONS affected by a peripheral injury, they and the circuits they form The presence of M-like currents sensitive to muscarine and sub- become hyperexcitable amplifying subsequent afferent input stance P modulation was fi rst reported in cultured spinal neurons abnormally. There is only one published study trying to evaluate using electrophysiological techniques (Nowak and Macdonald, possible changes in spinal Kv7 channel function as a consequence 1982, 1983a,b). Then, functional M-like currents were reported in of peripheral neuropathy (Passmore et al., 2003). This electrophysi- Laminae II–IV dorsal horn neurons (Murase et al., 1986). More ological study examined the responses of dorsal horn neurons to recently, retigabine was shown to produce signifi cant dose-related electrical and natural stimulation in rats that had undergone an inhibition of extracellular responses recorded from dorsal horn experimental neuropathy. The authors showed that retigabine pro- neurons in an in vivo rat preparation (Passmore et al., 2003). In duced similar depressant actions on the excitability of neurons in this study, retigabine potently suppressed nociceptive processing both naïve and treated animals. These results suggest that spinal and “wind up” in response to electrical stimuli. Wind-up consists expression of Kv7 channels is maintained after peripheral injury of a progressive increment of spike counts in response to repeti- and that these channels are still capable to infl uence excitability tive stimulation but only when nociceptive afferents are activated under the new conditions. (Herrero et al., 2000). We confi rmed these results in an in vitro In agreement with the previous results, unpublished observa- preparation of the rat spinal cord and showed that XE991 pro- tions from our laboratory indicate that Kv7.5 expression in the duced opposite effects and blocked the effects of retigabine (Rivera- spinal cord is not essentially altered by peripheral nerve injury. The Arconada et al., 2004). With intracellular recordings, we observed only apparent change is an increase in the percentage of superfi cial

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dorsal horn neurons expressing Kv7.5. An increase of functional Oral administration of retigabine to SNL rats reduced signs of M currents would contribute to stabilize neurons rather than to mechanical allodynia (measured as threshold for paw withdrawal). make them more excitable and therefore, if anything, this may In this case, retigabine was more potent than gabapentin (Dost be a homeostatic reaction to compensate for other sources of et al., 2004). This reduction was dose-dependent and reversed by excitability. co-administration of linopirdine indicating a specifi c action on Altogether, the data indicate that Kv7 channels are still present Kv7 channels. In these experiments, retigabine also reduced signs of in the spinal cord after neuropathy and may become valuable tar- heat hyperalgesia (measured as withdrawal latency to a heat source) gets for the control of neuronal excitability even in conditions of although reversal experiments were not performed. chronic pain and sensitization of the spinal cord. In a different experiment (Blackburn-Munro and Jensen, 2003), comparable oral doses of retigabine did not modify mechanical M CURRENT MODULATION IN BEHAVIORAL MODELS OF allodynia in CCI or SNI rats but reduced mechanical hyperalgesia NEUROPATHIC PAIN (assessed as withdrawal time to a pin-prick stimulus). In addition, Neuropathic pain patients develop multiple and complex symp- retigabine reduced cold allodynia (assessed as withdrawal time to toms likely refl ecting a variety of underlying physiopathological application of an ethyl chloride spray) in CCI rats although reversal mechanisms. However, it is generally admitted that neuropathic experiments were not reported. Unfortunately, this later effect was conditions involve damage to peripheral or central components of not observed in SNI rats. It is important to note that ataxic effects the nociceptive system leading to hyperexcitability of the system of retigabine appeared at doses about twofold greater than those itself. In peripheral nerves, spontaneous ectopic activity (attrib- required for their analgesic effects. uted to redistribution of K+ and Na+ channels, Devor et al., 1993; Other experiments have been performed with Kv7 openers using Rasband et al., 2001) is related to positive symptoms of neuropathy other pain models like intraplantar injection of capsaicin or forma- (Bostock et al., 2005; Devor, 2006; Ochoa et al., 2005; Orstavik et al., lin (Blackburn-Munro and Jensen, 2003; Passmore et al., 2003). In 2003). Hence, anticonvulsant/antiepileptic drugs such as gabap- both experiments oral administration of retigabine reduced signs entin and pregabalin have become an important option to treat of pain and these effects were antagonized by co-administration of neuropathic pain. Unfortunately, none of these drugs are 100% XE991. One more experiment has been performed using a model effective and their use is often associated with limiting and adverse of muscular pain which involves intramuscular injections of acidic side effects (Sindrup and Jensen, 1999); consequently, the search saline. In this case intraperitoneal administration of both retigabine for novel therapeutic targets is still compelling. and fl upirtine were shown to reduce signs of pain (Nielsen et al., The channel opener retigabine has been proven effective in 2004). various seizure models (Wuttke and Lerche, 2006) and it is in Signs of cognitive distress or spontaneous pain may accompany phase III clinical testing to treat pharmaco-resistant focal epilep- neuropathic syndromes (Andersen and Tufi k, 2003; Djouhri et al., sies at present (Bialer et al., 2009). Since Kv7 channel openers can 2006; LaBuda and Fuchs, 2000; Monassi et al., 2003; Pedersen and increase the stability of peripheral and central components of the Blackburn-Munro, 2006). In this regard, retigabine has been shown nociceptive system, they are potential targets of interest for neu- to reduce anxiety in models of unconditioned anxiety but not in ropathic pain. While there is a large body of experimental data on models of conditioned anxiety (Korsgaard et al., 2005; Munro and the effects of anticonvulsant drugs in the treatment of neuropathic Dalby-Brown, 2007). Mood modulation could be an additional pain (Blackburn-Munro and Erichsen, 2005), there is still sparse mechanism for Kv7 channel openers to improve neuropathic pain and at times puzzling data on the outcome of Kv7 modulation in conditions. this scenario. Data with new Kv7 channel openers on models of The general outcome of behavioral experiments is that open- neuropathic pain (like ICA-27243, fl upirtine or acrylamide) has ing of Kv7 channels tends to reduce signs of pain in models of been published only in abstract form (see Wickenden et al., 2004 neuropathy. This is hopeful; however, more experiments will be for a review) and only two full papers have been published with required to clarify present discrepancies and to establish the pain original data on the effects of retigabine on models of neuropathy conditions more susceptible to improve with retigabine and other (Blackburn-Munro and Jensen, 2003; Dost et al., 2004). Retigabine Kv7 channel openers. has been tested in three different models: spinal nerve ligation (SNL, Kim and Chung, 1992), chronic constriction injury (CCI, CONCLUDING REMARKS Bennett and Xie, 1988) and spared nerve injury (SNI, Decosterd and Although we may be looking at early times of research on the via- Woolf, 2000). The SNL model involves the ligation or sectioning of bility of M current modulation as a strategy for pain therapy, an a dorsal root, the CCI model uses loose ligatures around the sciatic emerging body of interesting and promising data is already avail- nerve and the SNI model sections the tibial and common peroneal able. Unfortunately many questions remain unanswered. branches of the sciatic nerve leaving the sural nerve intact. All three It is now very clear that functional M currents are present models have in common the fact that they produce a partial nerve in primary afferents and spinal neurons, both key elements for injury leaving extensive skin areas supplied by intact fi bers and nociceptive processing. In addition, they may be present in other therefore responsive to natural stimulation. Upon stimulation of areas related to pain processing. Are these channels involved in these areas, allodynia and hyperalgesia are commonly observed. the generation or maintenance of sensitized states leading to A feature that may differentiate between the models is the con- lasting pain? On the basis of existing data, it seems possible that tribution of intact and damaged fi bers to the observed symptoms endogenous modulation of the M current in dorsal root gan- (Campbell and Meyer, 2006). glion cells contributes to make sensory fi bers more excitable

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after peripheral infl ammation (Crozier et al., 2007; Linley et al., may benefi t from the development of Kv7 openers which do not 2008). However, this working hypothesis and its application to cross the blood brain barrier or compounds for topical applica- neuropathy will require further validation. On the other hand, tion that can be directly applied over the damaged nerves and be there is no evidence supporting a contribution of spinal M cur- absorbed through the skin. Data from spinal cord research draws rents to the initiation of central sensitization. A decreased expres- a much more complicated picture. M currents are present in most sion of the channel or the functional current in the spinal cord spinal neurons (sensory and motor) and other areas of the nerv- could be interpreted as an active mechanism leading to increase ous system. Therefore, compounds designed to act at this level the excitability of spinal neurons and therefore as a contribu- may show little selectivity for pain processing. A great amount of tion towards maintaining spinal sensitization. Such decreased information will be required to refi ne centrally acting Kv7 open- expression or function has not been found (see ‘M currents, the ers. Key data in this process are detailed maps of the expression spinal cord and neuropathy’). of individual Kv7 subunits by neurons of the nociceptive system Given this extensive expression of Kv7 channels in the noci- and the development of subunit-specifi c openers. However, as ceptive system which persists after injury, what is the interest with many other receptors and ion channels, there is no guarantee of Kv7 channels as targets for neuropathic pain? The outcome that a particular Kv7 subunit is central to pain processing during of behavioral studies is scarce but positive. Data available so far neuropathic pain. indicate that potentiation of M currents tend to reduce signs of pain in neuropathic animals. Basic research on primary afferents ACKNOWLEDGMENTS suggests that the site of injury at the periphery may be a particu- Authors are supported by the Spanish Ministry of Science and larly interesting point for the action of modulators (Lang et al., Education (Grant No. SAF 2006-03044) and the Local Government 2008; Roza and Lopez-Garcia, 2008). Some forms of neuropathy of Madrid (Grant No. S-SAL 0305-2006).

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