Expert Opinion On Therapeutic Targets Please download and read the instructions before proceeding to the peer review Are BK (big potassium) -type Ca2+ -activated potassium channels a viable target for the treatment of epilepsy? For Peer Review Only Journal: Expert Opinion On Therapeutic Targets Manuscript ID: EOTT-2014-0274.R1 Manuscript Type: Review BK potassium channels, Epilepsy, Seizures, BK channel modulators, Keywords: Antiepileptic drug (AED) URL: http://mc.manuscriptcentral.com/eott Email: [email protected] Page 1 of 48 Expert Opinion On Therapeutic Targets 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 The BK channel protein is a multimeric structure (homotetramer) composed of four identical pore-forming α 29 subunits (BKα), encoded by a single gene (Slo1, KCNMA1). Each BKα has seven trans-membrane segments 30 (S0-S6) and a large intracellular C-terminus region. The BKα protein shows three main structural domains, 31 each with a distinct function. The voltage-sensing domain (VSD) is located within the S1-S4 trans- 32 membrane segments, able to sense membrane potential. The S5-S6 segments form the pore gate domain 33 (PGD) including the activation gate, which controls K+ flux through the channel. The S6 segment serves as 34 the major structural determinant for the channel gate. The primary voltage sensor is located on the S4 35 helix, which contains many positively charged residues, but only one of these participates in voltage sensing. The membrane-spanning domains VSD and PGD, interact through the S4-S5 linker and S6, and the 36 hydrophobic segment (S0) leads to an extracellular NH2 terminus. The third domain is the cytosolic domain 37 (CTD), which enables the channel to respond to changes in [Ca2+]i and other stimuli. The intracellular C- 38 terminus has two tandem RCK (regulator of K+ conductance) domains, RCK1 and RCK2, folded tightly 39 against each other. Channel sensitivity to Ca2+ is determined by a gating ring of eight RCK domains from 40 the four assembled α subunits; this gating ring is subjected to an expansion during channel gating. RCK2 41 domains have an aspartate-rich region that forms the ‘Ca2+ bowl’, showing a high Ca2+ affinity. A low- 42 affinity Ca2+ recognition site has also been identified within the RCK1 domain, where another high-affinity Ca2+site is also present. RCK1 also mediates the channel’s sensitivity to Mg2+, Zn2+and Cd2+. The four β- 43 subunits (β1-4) are encoded by a specific gene KCNMB1-4 (human) or kcnmb 1-4 (mouse). These BK 44 channel subunits have two transmembrane domains (TM1 and TM2) connected by a large loop on the 45 extracellular side. β-subunits also show an intracellular N-terminus and C-terminus. Each β-subunit is 46 located between two adjacent α-subunits. γ subunits contain a single transmembrane domain, an N-terminal 47 extracellular LRRD (leucine-rich repeat domain), and a short C-terminal tail. 48 389x245mm (72 x 72 DPI) 49 50 51 52 53 54 55 56 57 58 59 60 URL: http://mc.manuscriptcentral.com/eott Email: [email protected] Expert Opinion On Therapeutic Targets Page 2 of 48 1 2 3 4 5 6 2+ 7 Are BK (big potassium)-type Ca -activated potassium channels a viable target for the 8 treatment of epilepsy? 9 10 11 12 Abstract 13 14 Introduction : BK (bigFor potassium) channelsPeer are Ca 2+ -activatedReview K + channels widely Only expressed 15 16 in mammalian cells. They are extensively distributed in the CNS, the most abundant level 17 18 being found in brain areas largely involved in epilepsy, namely cortex, hippocampus, 19 20 piriform cortex, and other limbic structures. BK channels control action potential 21 22 shape/duration, thereby regulating membrane excitability and Ca 2+ signalling. 23 24 Areas Covered: The potassium channel superfamily represents a rich source of potential 25 26 targets for therapeutic intervention in epilepsy. Some studies have identified alterations in 27 28 BK channel function, therefore, supporting the development of drugs acting on these 29 30 channels for epilepsy treatment. 31 32 Expert Opinion : The actual sketch is intriguing and controversial, since mechanisms altering 33 34 the physiological role of BK channels leading to either a loss- or gain-of-function have both 35 been linked to seizure onset. Not many studies have been performed to unravel the efficacy 36 37 of drugs acting on these channels as potential antiepileptics; however, paradoxically, efficacy 38 39 has been demonstrated for both BK channel openers and blockers. Furthermore, their 40 41 potential usefulness in preventing epileptogenesis has not been investigated at all. Substantial 42 43 data on risks and benefits of modulating these channels are urgently needed to draw a 44 45 definitive conclusion on whether BK channels are a viable future target for the treatment of 46 47 epilepsy. 48 49 50 51 Keywords: BK potassium channels; Epilepsy; Seizures; BK channel modulators; 52 53 Antiepileptic drug (AED). 54 55 56 57 58 59 1 60 URL: http://mc.manuscriptcentral.com/eott Email: [email protected] Page 3 of 48 Expert Opinion On Therapeutic Targets 1 2 3 4 5 6 7 8 9 Highlights Box 10 11 • BK channels control action potential shape and duration, thereby regulating 2+ 12 membrane excitability and Ca signalling; 13 14 • MechanismsFor altering the Peerphysiological role Reviewof BK channels leading toOnly either a 15 loss- or gain-of-function have both been linked to seizure onset; 16 • 17 Efficacy has been demonstrated for both channel openers and blockers in 18 preventing seizures; 19 • 20 BK channels might also be involved in epileptogenesis, however, no studies on the 21 efficacy of BK channel modulators in preventing epileptogenesis have been 22 performed so far; 23 • 24 The molecular structure of BK channels is described and allows the possibility of several target sites for potential pharmacological modulation. 25 26 27 28 List of abbreviations 29 [Ca 2+ ] = Calcium concentration 30 2+ 31 [Ca ]i = Intracellular calcium concentration 32 AEA = N-arach-idonylethanolamine 33 AED = Antiepileptic drug AHP = afterhyperpolarizationAfterhyperpolarization 34 Aps APs = Action potentials 35 4-AP = 4-Aminopyridine 36 ASN = Asparagine 37 Asp = Aspartate 38 ATP = Adenosine tri-phosphate 39 BK, BKCa, MaxiK, Slo1, KCa1.1, KCNMA1 = Big potassium channel or Big conductance 40 calcium-activated potassium channel 41 BKα = α subunit of BK channel 42 C-linker = Carboxy linker 43 C-terminus = Carboxy terminus 44 CA = Carbonic anhydrase 45 CA1 = Cornus ammonis 46 Ca 2+ = Calcium ions 47 Ca v = Calcium channel 48 cAMP = Cyclic adenosine monophosphate 49 CB = Cannabinoid receptors 50 CBD = Ccannabidiol 51 CBDV = Cannabidivarin 2+ 52 Cd = Cadmium ions 53 cGMP = cyclicCyclic -guanosine monophosphate 54 CNS = Central nervous system 55 56 57 58 59 2 60 URL: http://mc.manuscriptcentral.com/eott Email: [email protected] Expert Opinion On Therapeutic Targets Page 4 of 48 1 2 3 4 5 6 7 COX-2 = Cyclooxygenase-2 8 cryo-EM = cryo-Electron Microscopy CTD = Cytosolic domain 9 DHA = docosahexaenoic Docosahexaenoic acid 10 dSlow = drosophila Drosophila Slowpoke 11 ER = Endoplasmic reticulum 12 fAHP = fast afterhyperpolarization 13 GABA = γ-Aminobutyric acid 14 GH3 = Rat pituitary cellsFor Peer Review Only 15 Glu = Glutamate 16 GMP = Guanosine monophosphate 17 H+= Hydrogen ions 18 hSlow = human Slowpoke 19 IK = Intermediate potassium channel or Intermediate conductance calcium-activated 20 potassium channel 21 IP 3 = Inositol tri-phosphate 22 K+= Potassium ions 23 KCa = Calcium-activated potassium channel 24 Kv or KCNQ = Potassium channel 25 LRRCs = Leucine-rich repeat-containing subunits 26 mAHP = medium afterhyperpolarization 27 MAPK = Mitogen-activated protein kinase 28 MES = Electroshock-induce seizures 2+ 29 Mg = Magnesium ions 30 mitoBK = mitochondria Big Potassium channel 31 ms = millisecond mSlow = mice Slowpoke 32 + 33 Na = Sodium ions 34 Na v = Sodium channel NE = Nuclear envelope 35 NH or N terminus = Amino terminus 36 2 NMDA = N-methyl-D-aspartate 37 NO = Nitric oxide 38 PEA = N-palmitoylethanolamine 39 PGD = Pore gate domain 40 PI 3-kinase = Phosphoinositide 3-kinase 41 PKA = Protein kinase A 42 PKC = Protein Kinase C 43 PPAR-α = Peroxisome proliferator-activated receptor-alpha 44 PTZ = Pentylenetetrazole 45 RCK = Regulator of K + Conductance 46 ROS = Reactive oxygen species 47 s = second 48 S = trans-membrane segment 49 sAHP = slow afterhyperpolarization 50 SK = Small potassium channel or Small conductance calcium-activated potassium channel 51 Slo = Slowpoke 52 STREX = Stress axis hormone-regulated exon 53 TM = Transmembrane domain 54 TRPV = Transient receptor potential vanilloid 55 56 57 58 59 3 60 URL: http://mc.manuscriptcentral.com/eott Email: [email protected] Page 5 of 48 Expert Opinion On Therapeutic Targets 1 2 3 4 5 6 VSD = Voltage-sensing domain 7 2+ 8 Zn = Zinc ions ZNS = Zonisamide 9 τ = Time constant 10 11 12 13 14 1. Introduction For Peer Review Only 15 16 Calcium-activated potassium channels (KCa) are a large and diversified family of ion 17 2+ 2+ 18 channels that transduce increases in intracellular Ca ([Ca ]i) into changes in membrane 19 20 potential (hyperpolarization) that can then influence the duration and frequency of action 21 22 potentials (APs) in excitable cells (both pre- and postsynaptically) and thus exert an important 23 24 influence on their functional properties. According to their single channel conductance, Ca 2+ - 25 26 activated K + channels can be divided into three subfamilies: small conductance (SK: 2-25 pS), 27 28 intermediate conductance (IK: 25-100 pS) and large conductance (BK: 100–300 pS) subtypes; 29 1-4 30 each subgroup also exhibit distinct pharmacological and biophysical characteristics . In 31 2+ + 32 addition to their important regulatory roles, Ca -activated K channels also have an important 33 5-7 34 potential as targets for novel therapeutic drugs in health and disease .