Role of calcium-activated potassium channels in neuronal pacemaker activity

Nancy DONG,Zhong-ping FENG

（Department of Physiology,University of Toronto,1 King′s College Circle, Toronto,Ontario,Canada M5S 1A8）

·REVIEW·

Role of calcium-activated potassium channels in neuronal pacemaker activity

Nancy DONG,Zhong-ping FENG

（Department of Physiology,University of Toronto,1 King′s College Circle, Toronto,Ontario,Canada M5S 1A8）

Spontaneous rhythmic activity of pacemaker neurons in the central nervous system underlies fundamental neurological processes such as locomotion,cognition and circadian rhythm. Among the wide range of ion channels required for its generation,the Ca2+-activated K+(KCa)channels play a prominent role in maintaining physiologically-relevant frequency and pattern of pacemaker activity. Much of our understanding of the functions of KCachannels in pacemaker neurons have been derived from pharmacological studies using channel modulators,such as iberiotoxin and apamin. Despite the significant advances made,recent studies have painted an increasingly complex picture of the effects of widely used KCachannel modulators on unintended targets that may confound our under?standing of their functions.In this review,we discussed the utility and shortcomings of the KCachannel modulators,and highlighted the significance of these findings,because the KCachannel modulators have been used in early clinical trials to treat disorders ranging from Parkinson disease to alcoholism.

potassium channels,calcium-activated;pacemaker neurons;rhythmic activity;channel modulators

Pacemaker neurons are capable of generating rhythmic activity in the absence of external inputs[1].They play a key role in many neurological functions,including locomotion,cognition and circadian rhythm[2].The computational advantage of spontaneous firing lies in that both excitatory and inhibitory synaptic inputs can be coded in a timely fashion in changes in the frequency and/or pattern of rhythmic activity.Therefore,the ability to maintain stable and precise rhythmic activity is fundamental to the physiological functions of pacemaker neurons.

Pacemaker activity arises from an intricate interplay between the complement of ion channels present on the pacemaker neuron membrane, including hyperpolarization cyclic nucleotide(HCN)-activatedchannels[3],Na+leakchannels(NALCN)[4], voltage-gated Ca2+and Na+channels[5],and Ca2+-activated K+(KCa) channels[6]. KCachannels contribute to the repolarization and after-hyperpo?larization(AHP)phases of the action potential, thereby providing the negative feedback mechanism that stabilizes rhythmic activity in pacemaker neurons.Pharmacologicalmodulators ofKCachannels,such as iberiotoxin(IbTX)and periciazine (apamin),have been instrumental in elucidating the physiological functions of the large-conduc?tance(BK) and small-conductance(SK) KCachannels as regulators of pacemaker neuron activity[6]and as potential therapeutic targets in the treatment of neurological disorders[7-8].The goal of this article is to discuss the utility and potential caveats of commonly used pharmaco?logical agents in investigating the physiological function and therapeutic potential of KCachannels as regulators of pacemaker neuron activity.

1 LARGE-CONDUCTANCE KCaCHANNELS

1.1Structure and biophysical properties of BK channels

BK channels are composed of both a tetra?meric α pore-forming subunit[9]and modulatory β subunits[10].Each monomer of the α-subunit, encodedbytheSlo1gene,consistsofan N-terminal transmembrane pore forming region (S0-S6)and a C-terminal cytoplasmic regulatory domain(S7-S10).The S0 domain is required for interaction with β-subunits,which greatly diversify the channel properties.The S1-S4 domains make up the voltage sensor,by virtue of charged amino acid residues in the S2-S4 domains[11-13]. The P-loop is likewise found in between the S5 and S6 domains,and together with the other three subunits to form the pore region that con?tains the signature K+selectivity GYG motif and binding sites for the pore blockers charybdotoxin (ChTX)and IbTX[14-15].The C-terminal cytoplasmic domain can be divided into two regulator of K+conductance domains(RCK1 and RCK2)that each contain one low affinity Ca2+binding site[16-17]. The C-terminaldomains of all four subunits come together to form a″gating ring″[18]that makes Ca2+-dependent activation of BK channels possible.Multiple promoters and splicing variants of the subunits have been identified to give rise to diversity in the structure and properties of the pore forming domain[19].

The properties of a BK channel can be wideranging depending on the β-subunits associated with it.Currently,four β-subunits have been cloned[20-23]：β1is found in the smooth muscles,β2and β3specifically in neurons,and β4in the brain. The β-subunits associate in equal stoichiometric ratio with monomers of the α-subunits via the S0 segment to significantly modify the gating[24-25], pharmacological[26],and kinetic properties of the channel[27].For example,whereas BK channels containing the α-subunit alone or associated with the β1or β4-subunit do not inactivate,those with β2and β3-subunit show rapid inactivation[22-23].There?fore,careful biochemical characterization of the BK channels under examination is imperative before proceeding onto pharmacological studies.

BK channels activate rapidly in the coincident presence of membrane depolarization and an intracellular Ca2+concentration＞10 μmol·L-1[28]. The ensuing large K+current rapidly repolarizes the action potential and hyperpolarizes the membrane potential by mediating the fast component of the AHP(fAHP).The feedback loop is terminated with equal rapidity when BK channels are deactivated by membrane hyperpolarization and deactivation of voltage-gated Ca2+channels.

1.2 Pharmacological modulators of BK channels

As the structure and pharmacological prop?erties of BK channel modulators have been extensively reviewed elsewhere[29-30],only the most commonly used compounds in studying pacemaker neurons will be briefly introduced here.The earliest generation of BK channel blocker is ChTX,a potent yet largely non-selective K+channel blocker that is derived from the venom of the scorpionLeiurus quinquestriatus[31].It is now rarely used due to its additional activity inhibiting the voltage-gated K channels and inter?mediate-conductance KCa(IK)channels.

Currently two BK channel blockers have been used extensively(Tab.1).IbTX,the most commonly used BK channel blocker,was later isolated from the venom of another species of scorpionsButhus tamulus[32].Like ChTX,IbTX acts by binding to the external opening of BK channels.However,the latter exhibits higher affinity and selectivity for BK channels than the former,possibly due to slight differences in the amino acid make-up and resultant charge[32-33]. One potentially serious pitfall of IbTX is that β4-subunit-containing BK channels are shown to be resistant to it.

The tremorgenic indole alkaloid paxilline, produced byPenicillium paxilli,is the most widely used non-peptide BK channel blocker,due to itshigh potency,selectivity and reversibility of action[53]. Unlike ChTX and IbTX,paxilline acts on the αsubunit from the cytoplasmic side[54].The recently

Tab.1 Pharmacology of KCachannel blockers commonly used in studies of central pacemaker neurons in vitro and in vivo

isolated non-peptidergic BK channel blocker 1-〔1-hexyl-6-(methyloxy)-1H-indazol-3-yl〕-2-methyl-1-propanone(HMIMP)holds considerable promise, as it inhibits both the IbTX-sensitive and-resistant BK channel isoforms at nanomolar range and does not appear to affect human voltage-gated Na+,Ca2+and K+channels[55].

While activators of BK channels have long been identified,such as the benzoimidazolones NS004 and NS1619,their usefulness is limited because of their poor potency and selectivity[56-57]. Furthermore,these compounds are designed to act at membrane potentials~50-100 mV,which are more positive than the physiological range[58]. To address these shortcomings,the Hollywood group[59]synthesized a family of anthraquinone analogues,named GoSlo-SR,that are capable ofactivating BK channels atphysiological membrane potentials.

1.3 BK channels regulate central neuronal activities

As BK channel activity is time-locked to the action potential,itis particularly suited to shaping the spike profile.Indeed,in excised patch recordings of cerebellar Purkinje neurons, a significant amount of BK current is activated during action potential-like waveforms[34].IbTX is the most widely used tool to study the role of BK channels in regulating rhythmic firing frequency in several central pacemaker neuron populations, though with varied results.Currently,the best understood system is arguably the suprachias?matic nucleus(SCN),which is the central circadian pacemaker of the mammalian brain.Spontaneous firing frequency of the SCN neurons is modulated in a diurnal fashion to mark the day/night cycle,i.e.high during the subjective day and low during the subjective night[60].Downstream of the circadian clock genePer1[37],BK channel expression andactivity in SCN neurons peak during the middle of the night phase in order to suppress the frequency of spontaneous firing[38].This circadian pattern of rhythmic activity is blunted when BK channel activity is inhibited by IbTX[38],a result that is replicated in a global BK channel knock?out model[61].

A more complex picture is seen in the cerebellarPurkinje neurons,which highlights potential caveats of IbTX that should be taken into consideration when interpreting findings.In slice recordings,these neurons exhibit high frequency spontaneous firing(~40-50 Hz)[34].As the sole output neurons of the cerebellar cortex,the Purkinje neurons integrate a myriad of sensory, cortical and vestibular information needed to guide and coordinate voluntary motor behaviour[62]and encode them with high fidelity in the rate and precision of their firing activity.The discharge frequency is found to alternate over a wide range between rates higher and lower than the resting frequency in a consistent temporal relationship with the movement cycle[63].Indeed,recent studies have shown that the firing frequency is tightly controlled on the scale of milliseconds[64].Consistent with the role of BK channels in contributing to the fAHP,in vitrorecordings from acute cerebellar slices show that IbTX reduced the AHP[41]and increased the spontaneous firing activity[34,44].

Paradoxically,Purkinje neurons of global BK channel knockout animals exhibit reduced spon?taneous activity[65].While developmental compen?sation in the knockout animals is a possibility,it may be prudent to employ multiple BK channel blockers to control for potential off-target effects. Interestingly,the abnormal Purkinje cell firing pattern and ataxic behaviour in BK channel knockout mice were reproduced in wildtype mice byin vivomicroinjection of paxilline into the vermis[66],suggesting that paxilline may be an effective alternative to IbTX.On a related note, an IbTX-resistant but 1-ethyl-2-benzimidazolinone (1-EBIO)-sensitive BK current has recently been identified in Purkinje neurons[67].IbTX-and ChTX-resistant BK channels contain the β4-subunit, whose extracellular loop results in a~1000 fold decrease in toxin association,rendering them insensitive to nanomolar concentrations of these blockers that are normally sufficient to inhibit BK channels[23].The β4-subunit mRNA is abundant in the mammalian brain[23],suggesting that effects of IbTX may not be entirely attributable to inhibition ofBK channels.Furtherinvestigations are required to provide additional evidence.

1.4 Therapeutic potential of BK channels

From the evidences summarized above,BK channels are potential drug targets in the treatment of motor and circadian rhythm disorders,such as ataxia,narcolepsy and insomnia.However,rescue of BK channel function in disease states is hindered by the lack of effective channel activators, for the efficacy of GoSlo-SR compounds have yet to be testedin vivo.Conversely,the toxin BK channel blockers are poor drug candidates,for in addition to potential off-target effects,the pepti?dergic nature of ChTX and IbTX means that they are not active orally,have short half-lives and poor blood-brain permeability[29].Nevertheless, paxilline may be a promising target for future studies,as no major side-effects were detected in a study examining its efficacy as a systemicallyadministered anti-convulsant in rodents[68].

2 SMALL-CONDUCTANCE KCaCHANNELS

2.1Structure and biophysical properties of SK channels

Comparatively much more similar to voltagegated K+channels than BK channels,all three members of the SK family,SK1,SK2 and SK3, are homomeric tetramers of 6-transmembrane domain subunits[39].Thus it is the more surprising that SK channels are entirely voltage-independent, for the S4 segment contains only three positively charged residues as compared to many in most voltage-gated channels[69-70]. Instead,they are activated by submicromolar elevation in intracellular Ca2+by virtue of the constitutively bound calmodulin at the C-terminal calmodulin-binding domain (CaMBD)of each subunit[71-72].While the exactmechanism of gating is not yet clear,it is currently believed that Ca2+binding to calmodulin induces a conformational change in the arrangement of the tetramer that is translated into a mechanical force that opens the activation gate[73].This indirect coupling of Ca2+binding to channel opening results in slower activation kinetics as compared to BK channels,so that SK channels are fully open~5 ms after the action potential to mediate the medium AHP(mAHP)that lasts over hundreds of milliseconds[19].

2.2 Pharmacological modulators of SK channels

Excellent discussions of SK channel modulators can be found in two recent reviews[74-75],so only those most commonly used in studies of pacemaker neurons will be summarized here(Tab.1,channel blockers and Tab.2,channel activators).

The isolation of apamin,from the honeybee venom,as a selective blocker of SK2 and SK3 channels has been of immeasurable significance in elucidating the physiological function of SK channels[69,86].Initially believed to be a simple pore blocker based on the finding that several critical amino acid residues in the outer pore region are critical to sensitivity to apamin[40],there is recent evidence that apamin binding to the outer pore causes a conformational change in the selectivity pore that prevents K+conductance[87]. However,SK3 channels have been shown to be less sensitive than SK2 channels to blockade by apamin[88].It may therefore be advisable to prop?erly characterize the biochemical profile of the SK channel under examination and employ multiple pore blockers to control for theeffects of subtype specificity.To more selectively manipulate SK2 channels,the synthetic peptide toxin Lei Dab7 may be employed[43,52].

Tab.2 Pharmacology of KCachannel activators commonly used in studies of central pacemaker neurons in vitro and in vivo

Both 1-EBIO and chlorzoxazone(CZX)potentiate SK channel activity by increasing channel open probability[76].Interestingly,CZX is already an FDA-approved central muscle relaxant,making it an attractive drug candidate in modulation of SK channels in pacemaker neurons.Nevertheless, the lack ofspecificity/selectivity remains a challenge as 1-EBIO activates SK1,SK2,and SK3 channels equally[89]and both 1-EBIO and CZX activate IK channels,which are not expressed in neurons,in a concentration-dependent manner[76]. The most recently discovered activator,CyPPA, is ineffective against IK channels but cannot differentiate between SK2 and SK3 subtypes[80].

A recent study reported that the transient receptor potential melastatin member 7(TRPM7) channel is inhibited by SK channel activators derived from plant alkaloid quinine,including CyPPA,NS8593,SKA31 and UCL1684[81]. TRPM7channelsareubiquitouslyexpressed and implicated in a wide range of physiological functions including synaptic neurotransmission, intestinal peristalsis,regulation of vascular tone, bone growth and embryonic development[90],thus the effects of the plant alkaloid SK channel activators on related functions should be interpreted with caution.There is considerable promise in the CyPPA structural derivative NS13001,which is shown to be highly selective of the SK3 subtype and exhibit none of the off-target effects of CyP?PA in the micromolarconcentration range[43](Tab.2).

2.3 SK channels regulate pacemaker neuronal activities

In comparison with BK channels,the greater variety and selectivity of the SK channel modulators have allowed for better characterization of the role of SK channels in regulating pacemaker neuron activity.Through its contribution to the mAHP,SK channels control the firing threshold and thus regulate single spike in pacemaker cells.Its role is critical as a consistent mAHP after each spike ensures both that voltage-gated ion channels recover during each cycle and that cellular excitability remains low enough so random synaptic noises would not alter firing pattern.In addition to the frequency of firing,the precision/ regularity of rhythmic activity is another important component of the physiological functions of pace?maker neurons.

In cerebellar Purkinje neurons,precise rhythmic firing is required to accurately reflect the strength and timing of excitatory and inhibitory synaptic inputs that shape the final output of the cerebellum. In addition to BK channels,SK channels are highly expressed in cerebellar Purkinje neurons[91]. Pharmacological blockade of SK channels by apamin[6,35]or UCL1684[92]both resulted in decreased regularity of tonic spiking in Purkinje neurons.Enhancement of SK channel activity using 1-EBIO,riluzole or CZX restores regular Purkinje neuron firing in mice with either SK channel knockout[93]or with mutations in P/Q-type voltage-gated Ca2+channels[78],to which SK channels are closely coupled.

Spontaneously active dopaminergic(DA) neurons found in the substantia nigra pars com?pacta(SNc)and ventral tegmental area(VTA) exhibit two possible modes of firing,regular simple spiking and bursting.Changes in the firing patterns of these DA neurons impact the spatiotemporal profile of dopamine release in different regions of the brain,thereby affecting motor, motivation and memory processes in both physi?ological and pathological conditions,such as schizophrenia and Parkinson′s disease.Inhibition of SK channels in these neurons by apamin not only disrupts the precision of tonic firing but also causes transition to burst firing;and the effects of SK channel blockers were reversed by SK channel activators[46,48,94-95].Multiple subtypes of SK channels are expressed in these neurons,with SK3 channels being the most abundant[96].Deignan,et al[47]demonstrated SK2 and SK3 channels contribute differently to the pacemaker activity of SNc DA neurons,where the former is found primarily in the distal dendrites and regulates theprecision of spike firing,and the latter is mainly somatic and modulates the frequency of firing. While specific blockers to the channel subtypes are needed to elucidate the subtype-specific function of SK channels,it would be of interest to see if NS13001 can be used to selectively modulate SK3 channels in midbrain DA neurons.

Membrane depolarization and irregular firing due blockade of SK channels have also been reported in several other pacemaker neuron populations,such as the subthalamic nucleus[49], dorsalraphe nuclei[51]and externalglobus pallidus[50],suggesting that SK channels may play a conserved role as regulatorofpacemaker activity in the central nervous system.

2.4 Therapeutic potential of SK channels

Several SK channel modulators are currently showing promise as drug candidates.In the cerebellum,abnormal Purkinje neuron pacemaker activity and thus loss of its information encoding ability results in ataxia,a feature of many conditions characterized by gaitdisturbances,postural instability and degraded fine motor coordination. In rodent models of spinocerebellar ataxia types 2[43]and 3[97],episodic ataxia type 2[78,82],and ataxia caused by CACNA1A mutation[79],SK channel activators are shown to be able to restore regular Purkinje neuron pacemaker activity and improve motor performancein vivo.Most recently,a randomized,double-blind,placebo-controlled trial found that riluzole,another FDA-approved muscle relaxant and SK channel activator,can alleviate motor symptoms in some human patients of spinocerebellar ataxia or Friedreich′s ataxia with few side effects[85].

The basal ganglia are also critically involved in motor control,such that their neurodegeneration results in Parkinson′s disease,a progressive disorderthataffects generation ofvoluntary movements.Whereas neurons in both the SNc and subthalamic nucleus exhibit spontaneous rhythmic,single-spike activity in healthy subjects, burst-firing activity predominates in patients with Parkinson′s disease[98-99].Reduction in SK channel activity is hypothesized to underlie such a change,as they play a fundamental role in main?taining regular spiking behaviour and oppose the transition to burst firing[48-49].Consistent with such an idea,it has recently been shown that apamin alleviates some motor deficits in a rat model of Parkinson′s disease[42].

In addition to motor processes,midbrain DA neurons also play an important role in cognitive functions such as memory,reward and motivation. The KCNN3 gene,which encodes the SK3 channel,contains a polymorphic CAG repeat in the amino-terminal cording region whose length has been implicated in schizophrenia[100-101]and anorexia nervosa[102].SK channels may also be a drug target in the treatment of addiction,for it has recently been shown that the systemic administration of the FDA-approved muscle relaxant CZX reduces excessive alcohol intake in rats[7].

Nevertheless,the potential for widespread side-effects is a concern for systemically adminis?tered SK channel modulators.All three subtypes of SK channels are widely expressed throughout the brain[103-104]and the body,including vascular endothelium,skeletal muscle,smooth muscle, and cardiac myocytes[105].In addition,as mentioned above,the SK channel activators 1-EBIO and CZX also potentiate IK channel activity due to their structural similarities[76-77].While IK channels are not expressed in neurons,they are widely expressed in peripheral tissues,such as the colon,placenta,lungs and pancreas.Indeed, reported side effects of orally administered CZX and riluzole are mild but wide ranging,including nausea,diarrhea,hypertension,and somno?lence[106].Memory impairments are also possible, given studies showing that transgenic animals over-expressing SK2 channels exhibitimpaired performance in hippocampal-dependent memory tasks[107-108]and the systemic administration of CyPPA impairs encoding of object recognition memory in mice[109].

3 CONCLUSION

Pharmacologicalactivators and blockershave been instrumental in advancing our under?standing of the role of KCachannels as regulators of pacemaker neuron activity in the central nervous system.Nevertheless,as with all experimental approaches,knowledge of their limitations is necessary for proper interpretation of the findings. This is all the more important now as several of these compounds are being tested in human clinical trials.The discovery of increasingly more potent and selective KCachannel modulators holds great promise for unraveling the mechanism and physiological functions of pacemaker neuron activity in future studies.

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鈣離子激活鉀通道對(duì)起搏神經(jīng)元活性的作用

Nancy DONG，馮中平

（Department of Physiology，University of Toronto，1 King′s College Circle，Toronto，Ontario，Canada M5S 1A8）

中樞起搏神經(jīng)元的自發(fā)節(jié)律活動(dòng)是神經(jīng)功能的基礎(chǔ)，這些神經(jīng)功能例如體位移動(dòng)，晝夜節(jié)律和認(rèn)識(shí)知覺。神經(jīng)元的自發(fā)節(jié)律活動(dòng)的生成涉及多種離子通道，鈣離子激活鉀通道（KCa通道）在維持生理性起博頻率和規(guī)律中起著的突出的作用。根據(jù)對(duì)iberiotoxin和蜂毒明肽類的KCa通道調(diào)節(jié)藥物的藥理研究，我們對(duì)KCa通道在起搏神經(jīng)元功能中的作用的認(rèn)識(shí)獲得很大的提高。盡管近年的研究取得了顯著的進(jìn)步，鈣激活鉀通道調(diào)節(jié)劑的廣泛使用增加了非特異性藥物作用意想不到的復(fù)雜性。由于KCa通道調(diào)節(jié)劑已經(jīng)用于帕金森病和乙醇中毒等多種疾病的早期臨床試驗(yàn)治療，本文強(qiáng)調(diào)了這些藥物作用和不足在使用中的重要性。

鉀通道鈣激活；自發(fā)起搏神經(jīng)元；節(jié)律活動(dòng)；通道調(diào)節(jié)劑

馮中平，E-mail：zp.feng@utoronto.ca，Tel：+1（416）946-0671

2016-03-16 接受日期：2016-06-13）

R966

:A

:1000-3002-(2016)06-0627-13

10.3867/j.issn.1000-3002.2016.06.003

Biography:Nancy DONG,female,HBSc,graduate student, main research field is neuroscience,E-mail:nancy.dong@ mail.utoronto.ca

Zhong-ping FENG,Tel:+1(416) 946-0671,E-mail:zp.feng@utoronto.ca

（本文編輯：喬 虹）