Abstract
Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.
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References
Bezanilla F (2002) Voltage sensor movements. J Gen Physiol 120:465–473. doi:10.1085/jgp.20028660
Bracey K, Wray D (2006) Inherited disorders of ion channels. In: Voltage gated ion channels as drug targets, Wiley, NY
Chen J, Zou A, Splawski I, Keating MT, Sanguinetti MC (1999) Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of HERG potassium channels accelerate channel deactivation. J Biol Chem 274:10113–10118. doi:10.1074/jbc.274.15.10113
Craven KB, Olivier NB, Zagotta WN (2008) C-terminal movement during gating in cyclic nucleotide-modulated channels. J Biol Chem 283:14728–14738. doi:10.1074/jbc.M710463200
Cui J, Kagan A, Qin D, Mathew J, Melman YF, McDonald TV (2001) Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with KCNE2. J Biol Chem 276:17244–17251. doi:10.1074/jbc.M010904200
Gandhi CS, Isacoff EY (2002) Molecular models of voltage sensing. J Gen Physiol 120:455–463. doi:10.1085/jgp.20028678
Gomez-Varela D, de la Pena P, Garcia J, Giraldez T, Barros F (2002) Influence of amino-terminal structures on kinetic transitions between several closed and open states in human erg K+ channels. J Membr Biol 187:117–133. doi:10.1007/s00232-001-0156-4
Jiang Y, Lee A, Chen J, Cadene M, Chalt BT, MacKinnon R (2002) The open pore conformation of potassium channels. Nature 417:523–526. doi:10.1038/417523a
Jones PA, Tucker SJ, Ashcroft FM (2001) Multiple sites of interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2. FEBS Lett 508:85–89. doi:10.1016/S0014-5793(01)03023-X
Ju M, Wray D (2002) Molecular identification and characterisation of the human eag2 potassium channel. FEBS Lett 524:204–210. doi:10.1016/S0014-5793(02)03055-7
Ju M, Wray D (2006) Molecular regions responsible for differences in activation between heag channels. Biochem Biophys Res Commun 342:1088–1097. doi:10.1016/j.bbrc.2006.02.062
Ju M, Stevens L, Leadbitter E, Wray D (2003) The roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel. J Biol Chem 278:12769–12778. doi:10.1074/jbc.M212973200
Lee SY, Lee A, Chen J, MacKinnon R (2005) Structure of KvAP voltage-dependent K+ channel and its dependence on the lipid membrane. Proc Natl Acad Sci USA 102:15441–15446. doi:10.1073/pnas.0507651102
Lorinczi E, Napp J, Contretas-Jurado C, Pardo LA, Stuhmer W (2008) The voltage dependence of hEag currents is not determined solely by membrane-spanning domains. Eur Biophys J. doi:10.1007/s00249-008-0319-7
Milligan CJ, Wray D (2000) Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis. Biophys J 78:1852–1861
Mohapatra DP, Siino DF, Trimmer JS (2008) Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation of the Kv2.1 channel. J Neurosci 28:4982–4994. doi:10.1523/JNEUROSCI.0186-08.2008
Morais Cabral JH, Lee SL, Cohen BT, Chait BT, Li M, Mackinnon R (1998) Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. Cell 95:649–655. doi:10.1016/S0092-8674(00)81635-9
Occhiodoro T, Bernheim L, Liu JH, Bijlenga P, Sinnreich CR, Bader CR, Fischer-Lougheed J (1998) Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion. FEBS Lett 434:177–182. doi:10.1016/S0014-5793(98)00973-9
Pardo LA, Stuhmer W (2008) Eag1: an emerging oncological target. Cancer Res 68:1611–1613. doi:10.1158/0008-5472.CAN-07-5710
Scholle A, Zimmer T, Koopmann R, Engeland B, Pongs O, Benndorf K (2004) Effects of Kv1.2 intracellular regions on activation of Kv2.1 channels. Biophys J 87:873–882. doi:10.1529/biophysj.104.040550
Schonherr R, Heinemann SH (1996) Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J Physiol 493:635–642
Terlau H, Heinemann SH, Stuhmer W, Pongs O, Ludwig J (1997) Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment. J Physiol 502:537–543. doi:10.1111/j.1469-7793.1997.537bj.x
Viloria CG, Barros F, Giraldez T, Gomez-Varela D, de la Pena P (2000) Differential effects of amino-terminal distal and proximal domains in the regulation of human erg K(+) channel gating. Biophys J 79:231–246
Wang J, Trudeau MC, Zappia AM, Robertson GA (1998) Regulation of deactivation by an amino terminal domain in human ether-à-go-go-related gene potassium channels. J Gen Physiol 112:637–647. doi:10.1085/jgp.112.5.637
Wang J, Myers CD, Robertson GA (2000) Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels. J Gen Physiol 115:749–758. doi:10.1085/jgp.115.6.749
Wray D (2000) Ion channels: molecular machines par excellence. Sci Spectr 23:64–71
Yellen G (2002) The voltage-gated potassium channels and their relatives. Nature 419:35–42. doi:10.1038/nature00978
Yusaf SP, Wray D, Sivaprasadarao A (1996) Measurement of the movement of the S4 segment during the activation of a voltage-gated potassium channel. Eur J Phys 433:91–97. doi:10.1007/s004240050253
Zagotta WN, Olivier NB, Black KD, Young EC, Olson R, Gouaux E (2003) Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425:200–205. doi:10.1038/nature01922
Acknowledgments
This research was supported by the Biotechnology and Biological Sciences Research Council (Grant Ref; BB/C004922/1). Construction of the tetrameric cNBD model from the single homology model subunit was made by S.E.V. Phillips and C. Trinh; the model for relative orientations of subunits was by D. Elliott.
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Stevens, L., Ju, M. & Wray, D. Roles of surface residues of intracellular domains of heag potassium channels. Eur Biophys J 38, 523–532 (2009). https://doi.org/10.1007/s00249-009-0402-8
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DOI: https://doi.org/10.1007/s00249-009-0402-8