14-3-3h is a Binding Partner of Rat Eag1 Potassium Channels Po-Hao Hsu1¤, Shi-Chuen Miaw3, Chau-Ching Chuang1¤, Pei-Yu Chang1, Ssu-Ju Fu1¤, Guey-Mei Jow4, Mei-Miao Chiu1, Chung-Jiuan Jeng1,2* 1 Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, 2 Brain Research Center, National Yang-Ming University, Taipei, Taiwan, 3 Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan, 4 School of Medicine, Fu-Jen Catholic University, Hsin- Chuang, New Taipei City, Taiwan Abstract The ether-a`-go-go (Eag) potassium (K+) channel belongs to the superfamily of voltage-gated K+ channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3h, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3h was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3h led to a sizable suppression of rEag1 K+ currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3h failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3h binding may render a fraction of the channel locked in a non- conducting state. Together these data suggest that 14-3-3h is a binding partner of rEag1 and may modulate the functional expression of the K+ channel in neurons. Citation: Hsu P-H, Miaw S-C, Chuang C-C, Chang P-Y, Fu S-J, et al. (2012) 14-3-3h is a Binding Partner of Rat Eag1 Potassium Channels. PLoS ONE 7(7): e41203. doi:10.1371/journal.pone.0041203 Editor: Stuart E. Dryer, University of Houston, United States of America Received March 9, 2012; Accepted June 18, 2012; Published July 20, 2012 Copyright: 2012 Hsu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from National Science Council, Taiwan (NSC97-2320-B-010-020-MY3) and from the Aim for the Top University Plan, Ministry of Education, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cjjeng@ym.edu.tw ¤ Current address: Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan Introduction The ether-a`-go-go (Eag) potassium (K+) channel belongs to the EAG family of voltage-gated K+ (Kv) channels that comprises three gene subfamilies: eag, erg (eag-related gene), and elk (eag-like K+ channel) [1]. Like other Kv channels, a functional Eag K+ channel is a tetramer comprising four pore-forming subunits [2]. In mammals, two neuron-specific Eag subunit isoforms (Eag1 and Eag2) have been identified [3,4,5]. Results from in situ hybridiza- tion studies in rats have demonstrated that rat Eag1 (rEag1) and Eag2 (rEag2) K+ channel subunits are widely expressed in various regions of the brain [4,6]. Despite their abundant expression in the brain, the neurophys- iological role of Eag K+ channels remains obscure. One strategy to tackle this question is to identify potential Eag-interacting proteins. Epsin, a protein involved in endocytosis and cell cycle regulation, was previously shown to interact with rEag1, suggesting a func- tional link of the K+ channel to cell cycle-related signaling [7]. Calmodulin, a calcium-binding protein, has also been demon- strated to exert a calcium-dependent inhibitory effect on human Eag1 K+ channels [8,9]. Interestingly, Drosophila Eag K+ channels can directly interact with Ca2+/calmodulin-dependent protein kinase II (CaMKII) [10,11], which is an abundant enzyme in neurons that has been implicated to play a critical role in the modulation of synaptic plasticity [12,13]. In addition, Camguk, a membrane-associated guanylate kinase adaptor protein that associates with CaMKII [14], was found to promote the surface expression of Drosophila Eag [15]. It is still unknown, however, whether CaMKII and CASK/Lin-2 (the mammalian ortholog of Camguk) may also interact with and/or modulate the biophysical properties of mammalian Eag K+ channels. To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. By applying the yeast two- hybrid screening of a rat brain cDNA library, we have identified 14-3-3h as a binding partner of rEag1 K+ channels. In addition, we have employed biochemical, morphological, and electrophys- iological assays to characterize this novel protein interaction between 14-3-3h and rEag1 K+ channels. Materials and Methods Ethics Statement All animals were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). All proce- PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41203