14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels

Abstract

The ether-à-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 14-3-3θ, 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 14-3-3θ 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 14-3-3θ 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 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K + channel in neurons.

Structure of the voltage-gated K + channel Eag1 reveals an alternative voltage sensing mechanism

“…However, the intracellular vestibule formed by the CNBHD does not contain many negative charges as is found in many cation channels, which may explain the low conductance of these channels (8 pS) (Fig. S7) (33). Like previous CNBHD structures, a portion of the Eag1 sequence occupies the cyclic nucleotide-binding site, thus preventing cyclic nucleotide binding (Fig.…”

Ca2+‐binding protein centrin 4 is a novel binding partner of rat Eag1 K+ channels

“…Whole‐cell patch clamp in HEK293T cells was performed at 24–48 h post‐transfection as previously reported . Patch electrodes with a resistance of ~ 4 MΩ were filled with a solution containing (in m m ) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2.…”

“…The DupLEX‐A yeast two‐hybrid system (OriGene, Rockville, MD, USA) was employed to screen a rat brain cDNA library as previously reported . Briefly, the cDNA portion corresponding to rEag1 carboxyl‐terminal region was subcloned into the yeast expression plasmid pGilda, which was used as the bait to screen the library.…”

Integrate Omics Data and Molecular Dynamics Simulations toward Better Understanding of Human 14-3-3 Interactomes and Better Drugs for Cancer Therapy

“…Like most other 14-3-3s, 14-3-3ζis expressed abundantly in the brain, and its misregulation is often involved in the development and progression of neurological disorders such as Parkinson'sdisease (Hsu et al, 2012;Ding et al, 2013). This isoform may also play a role in other diseases, such as leukemia, by increasing cell survival and inhibiting the activity of proapoptotic proteins (Scheibner et al, 2012).…”

“…This isoform may also play a role in other diseases, such as leukemia, by increasing cell survival and inhibiting the activity of proapoptotic proteins (Scheibner et al, 2012). 14-3-3ζis also a direct binding partner of the Eag potassium channel in mammals, and significantly inhibited K + current in rats upon its overexpression (Hsu et al, 2012).…”