nucleotides have been shown not to modulate Eag channels [40]. Interestingly, previous studies based on homology modeling and mutation analyses suggest that significant domain interaction may exist between the PAS domain and the CNBHD [41], which is not inconsistent with our assertion that the PAS domain and the CNBHD region constitute the N- and the C-terminal 14-3-3h interaction sites, respectively. Further investigation will be re- quired to determine whether one 14-3-3h dimer per se could simultaneously interact with the PAS domain and the CNBHD region, which in turn may provide important structural insight into the intra- and inter-subunit organization of rEag1 K+ channels. Previously, over-expression of 14-3-3f was shown to enhance the membrane trafficking of TASK K+ channels [29]. Likewise, endogenous 14-3-3 proteins promote the surface expression of ATP-sensitive K+ channels [42]. Our biochemical analyses, however, showed that neither the total protein expression nor the surface trafficking of rEag1 channels was reduced in the presence of over-expressed 14-3-3h protein. Similar to our finding, it has also been demonstrated that in spite of a significant inhibition of Ca2+ extrusion function, over-expression of 14-3-3e did not affect the level of protein expression or the membrane targeting of recombinant Na+-Ca2+ exchanger [43]. Moreover, 14- 3-3e was known to augment the activity of the Herg channel, another member of the EAG K+ channel family, by altering the channel’s steady-state voltage dependence of activation [35]. In the current report, co-expression with 14-3-3h, however, failed to exert discernible effect on steady-state voltage dependence, gating kinetics, or single channel conductance of rEag1 channels. Notably, the modulatory effect of 14-3-3e required the phosphor- ylation of Herg channels [35](Fig. S1), whereas 14-3-3h appeared to interact with rEag1 in a phosphorylation-independent manner. We therefore propose that 14-3-3h binding may modulate the protein conformation of rEag1 such that a fraction of the K+ channel is virtually locked in a non-conducting closed or inactivated state. Clearly, more research is needed to understand the molecular nature of this 14-3-3h-induced conformation change. Nevertheless, we cannot rule out the possibility that 14- 3-3h may also interact with certain rEag1-modulating factor(s) in cells, thereby indirectly down-regulating the functional expression of rEag1 K+ channels. 14-3-3 proteins are thought to function as U-shaped dimeric proteins, either homodimers or heterodimers, with the N- and C- terminal helices of each monomer serving as the dimerization and the ligand-binding domain, respectively [24,44,45]. The interac- tion between 14-3-3h and rEag1 is unique in that the rEag1 protein displays a remarkable binding preference to 14-3-3h over other 14-3-3 isoforms (see Figure 2). By contrast, TASK K+ channels, N-type Ca2+ channels, and Na+-Ca2+ exchangers virtually interact with all isoforms of 14-3-3 [22,29,43]. Currently, there is no clear molecular determinant available to explain ligand discrimination among 14-3-3 isoforms [24]. One potential way to address this issue in the future is to determine whether various 14- 3-3h-containing heterodimers can efficiently interact with and differentially regulate the functional expression of rEag1 K+ channels. Despite their widespread expression in various regions, the precise neurophysiological functions of rEag1 K+ channels in the brain remain elusive. Our previous immunofluorescence studies in hippocampal and retinal neurons demonstrate that rEag1 channels display a broad localization pattern over the somatoden- References 14-3-3h Interacts with rEag1 dritic compartment, extending from somas to distal dendrites and postsynaptic membranes [16,46]. Similarly, in the current study we found that 14-3-3h and rEag1 exhibit significant subcellular co- localization pattern in neurons, including the synaptic region. These data are consistent with the idea that rEag1 K+ channels may contribute to the control of neuronal excitability over a wide range of subcellular compartment. Given our finding that 14-3-3h suppresses rEag1 K+ currents, these observations raise a possibility that 14-3-3 protein may influence the functional expression of rEag1 channel in specific subcellular compartments of neurons, a plausible idea worthy of future investigation. Since 14-3-3 proteins are abundantly expressed in the brain, it seems likely that the interaction between 14-3-3h and rEag1 is constitutive and possesses little chance for physiological regulation. On the other hand, recent proteomic analyses of transgenic mouse brain tissues revealed that 14-3-3 proteins are associated with numerous binding partners essential for synaptic signaling and structural modulation of dendritic spines [47]; for example, 14-3- 3f may regulate postsynaptic glutamate receptor signaling through its interaction with Homer 3, a glutamate receptor-associated scaffolding protein that is also known to form synapse protein complexes with other postsynaptic density proteins such as PSD- 95 [48,49]. Moreover, PSD-95-associated multi-protein complexes were further demonstrated to involve over a hundred proteins crucial for synaptic functions, including glutamate receptors, K+ channels, and scaffolding proteins [50]. It remains to be de- termined, therefore, whether the interaction between 14-3-3 and rEag1 may also be subject to the modulation by synaptic signaling processes involving the 14-3-3 protein family. Supporting Information Figure S1 Phosophorylation-dependent interaction of Herg with 14-3-3e. Herg was co-expressed with an empty vector or myc-tagged 14-3-3e in HEK293T cells. 24 hours after trans- fection, cells were left untreated, or were treated with 1 mM okadaic acid or staurosporine for 60 min. (Left panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti- phosphorylated Akt (pAkt) antibody. b-actin was run as a loading control. (Right panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or anti-Herg antibody. In the presence of the phosphatase inhibitor okadaic acid, enhanced immunopreciation efficiency was observed for Herg. By contrast, pretreatment with the protein kinase-inhibitor staurosporine led to a small but conspicuous decrease in 14-3-3e interaction. These co-immuno- precipitation data are representative of three independent experiments. (TIF) Acknowledgments We thank Drs. Chih-Yung Tang and Yi-Hsuan Lee for technical guidance and critically reading the manuscript. Author Contributions Conceived and designed the experiments: SCM CJJ. Performed the experiments: PHH PYC CCC SJF GMJ MMC. Analyzed the data: PHH PYC CCC SJF CJJ. Contributed reagents/materials/analysis tools: SCM GMJ CJJ. Wrote the paper: CJJ. 1. Warmke JW, Ganetzky B (1994) A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A 91: 3438–3442. 2. MacKinnon R (1991) Determination of the subunit stoichiometry of a voltage- activated potassium channel. Nature 350: 232–235. PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e41203