1 3 Differential expression of two-pore domain potassium channels in rat 4 cerebellar granule neurons 5 6 7 Paulina Burgos a Q1 , Rafael Zúñiga a , Pedro Dominguez a , Fernando Delgado-Lopez b , Leigh D. Plant c , 8 Leandro Zúñiga a,⇑ 9 a Centro de Investigaciones Médicas CIM, Escuela de Medicina, Universidad de Talca, Talca, Chile 10 b Escuela de Medicina, Universidad Católica del Maule, Talca, Chile 11 c Department of Biochemistry, Brandeis University, Waltham, USA 12 13 15 article info 16 Article history: 17 Received 26 September 2014 18 Available online xxxx 19 Keywords: 20 K2P channels 21 Two-pore domain potassium channels 22 Cerebellar granule neurons (CGNs) 23 24 abstract 25 Two pore domain potassium (K2P) channels are mostly present in the central nervous system (CNS) 26 where they play important roles in modulating neuronal excitability. K2P channels give rise to back- 27 ground K + currents (IK SO ) a key component in setting and maintaining the resting membrane potential 28 in excitable cells. Here, we studied the expression and relative abundances of K2P channels in cerebellar 29 granule neurons (CGNs), combining molecular biology, electrophysiology and immunologic techniques. 30 The CGN IK SO was very sensitive to external pH, as previously reported. Quantitative determination of 31 mRNA expression level demonstrated the existence of an accumulation pattern of transcripts in CGN that 32 encode K2P9 > K2P1 > K2P3 > K2P18 > K2P2 = K2P10 > K2P4 > K2P5 subunits. The presence of the major 33 K2P subunits expressed was then confirmed by Western blot and immunofluorescence analysis, 34 demonstrating robust expression of K2P1 (TWIK-1), K2P3 (TASK-1), K2P9 (TASK-3) and K2P18 (TRESK) 35 channel protein. Based, on these results, it is concluded that K2P1, -3, -9 and -18 subunits represent 36 the majority component of IK SO current in CGN. 37 Ó 2014 Published by Elsevier Inc. 38 39 40 41 1. Introduction 42 Two pore domain potassium channels (K2P) give rise to the leak 43 or background potassium currents which are voltage independent 44 and constitutively active in many excitable cells. In mammals, K2P 45 channels are formed from 15 different subunits, which are divided 46 in six subfamilies based on the structural and functional properties 47 [10,17,19]. Structurally, each K2P subunit contains four transmem- 48 brane domains and two pore forming domains in tandem [5,18,22]. 49 A functional channel is composed of two identical (homomeric) or 50 different subunits (heterodimeric), that each imparts different 51 functional properties to the channel [1,6,15,26,30]. The heterodi- 52 meric configuration of K2P channels increases the functional diver- 53 sity thereby conferring versatility and dynamic adaptation to fulfill 54 the physiological roles which are implicated [26]. 55 K2P channels are highly regulated by several molecules or stim- 56 uli such as kinases, phosphatases, lipids, G proteins, internal and 57 external pH [9,19]. Extracellular pH modulates K2P channels by 58 acting on the upper gate [16]. At the molecular level, the mecha- 59 nism that confer pH sensitivity has been identified for several 60 members of the K2P family [16]. In the case of the pH-sensitive 61 channels K2P1 (also called TWIK-1 or KCNK1), K2P3 (also called 62 TASK-1 or KCNK3) and K2P9 (also called TASK-3 or KCNK9), acidi- 63 fication blocks the pore of the channel by protonating a histidine at 64 position 98 for K2P3 and 9 and 122 for K2P1. These histidine 65 residues are located adjacent to the potassium selectivity sequence 66 in the first pore forming loop of each subunit of the channel 67 [23,27,29]. 68 In neurons, where K2P channels are highly expressed, the cur- 69 rents generated by these proteins have been well characterized 70 and are known as IK SO (for standing outward potassium current) 71 [21,32]. The IK SO current is strongly modulated by extracellular 72 pH [12,15,21]. And the inhibition of IK SO by acidosis has been asso- 73 ciated with an increased of excitability [24,25]. 74 CGNs are glutamatergic interneurons that provide an excitatory 75 input in the molecular layer of the cerebellum. The IK SO in these 76 cells has been correlated with the expression of four K2P genes: 77 K2P1, K2P10 (TREK-2, KCNK10), K2P3 and K2P9 [1,4,11,12,14,21, 78 26,28,31]. Of these subunits, K2P3, K2P9 and K2P1 can also com- 79 bine to form heterodimeric channels [1,6,15,26,30]. Furthermore, 80 the regulation of K2P1, K2P3 and K2P9 heterodimers has been http://dx.doi.org/10.1016/j.bbrc.2014.10.012 0006-291X/Ó 2014 Published by Elsevier Inc. ⇑ Corresponding author. E-mail address: lzuniga@utalca.cl (L. Zúñiga). Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 32837 No. of Pages 7, Model 5G 11 October 2014 Please cite this article in press as: P. Burgos et al., Differential expression of two-pore domain potassium channels in rat cerebellar granule neurons, Bio- chem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.10.012