The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

Mammalian Eag1 (Kv10.1) potassium (K+) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K+ channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels.

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“…Transient transfection was performed by the calcium phosphate method49. Cells were plated onto 6- or 12-well plates 24 hrs before transfection.…”

Section: Methodsmentioning

confidence: 99%

Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

Hsu

Yu²,

Fang

et al. 2017

Sci Rep

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“…To further understand the structural basis underlying 14-3-3 regulation of Cul7-mediated Eag1 degradation, we employed chimeric constructs of two distinct subtypes of the KCNH channel family: rat Eag1 (Kv10.1; KCNH1) and human Erg (hErg; Kv11.1; KCNH2) (Fig. 10 A) [ 38 , 39 ]. Figure 10 B depicts that replacing the CNBHD, but not the C-linker, of Eag1 with that of hErg prevented difopein from boosting Eag1 protein level.…”

Section: Resultsmentioning

confidence: 99%

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

et al. 2023

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Background Mutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis. Results In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins. Conclusion Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

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“…The clay particles (Fig. a) produce characteristic bonds associated with the stretching of SiO (1037 cm −1 ), and SiOAl (522 cm −1 ), and bonding of SiOSi at 463 cm −1 . Also, the modified clay shows the existence of some bonds due to the presence of 2‐BBI.…”

Section: Resultsmentioning

confidence: 99%

Anticorrosive performance of epoxy/modified clay nanocomposites

Mehrabian

Dariani

2017

Polymer Composites

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The effect of 2‐benzylbenzimidazole (2‐BBI) was evaluated as surface modifier on the performance of Na+‐Montmorillonite nanoparticles (Na+‐MMT) in epoxy‐based coatings. Four epoxy‐based samples were prepared including 0, 1, 2, and 3 wt% of 2‐BBI‐(Na+‐MMT). The intercalation behaviors of epoxy resin in presence of 2‐BBI‐(Na+‐MMT) were determined by X‐ray diffraction (XRD), and field emission scanning electron microscope (FE‐SEM) analysis was carried out to depict the dispersion of 2‐BBI‐(Na+‐MMT). The pertaining reaction bonds of the 2‐BBI and (Na+‐MMT) were represented via Fourier transform infrared spectroscopy (FTIR), and pull‐off test was used for measuring the adhesion strength of the coated samples. Corrosion resistance and thermal properties of the coated samples were measured by electrochemical impedance spectroscopy (EIS) and differential scanning calorimetry (DSC), respectively. On the basis of the EIS results, the coated samples with 3 wt% 2‐BBI‐(Na+‐MMT)/epoxy had the highest corrosion resistance after 120 days immersion in 3.5 wt% NaCl solution. Also, based on the Brusher–Kingsbury equation, the amount of water absorption was calculated, in which 3 wt% 2‐BBI‐(Na+‐MMT)/epoxy had only 1.92% water absorption while 3 wt% (Na+‐MMT)/epoxy had 82 wt%. The results showed that 2‐BBI can improve the dispersion of the Na+‐MMT in the epoxy matrix by entering into their galleries and providing better compatibility between the inorganic clay nanoparticles and polymeric matrix. This issue led to the enhancement of the thermal and barrier properties of 2‐BBI‐modified (Na+‐MMT)/epoxy nanocomposites rather than unmodified ones. POLYM. COMPOS., 39:E2134–E2142, 2018. © 2017 Society of Plastics Engineers

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The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

Abstract: Background: K ϩ channel subunits from different ether-à-go-go subfamilies cannot form heterotetramers.

Cited by 15 publications

References 42 publications

Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

Anticorrosive performance of epoxy/modified clay nanocomposites

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