The punctate localization of rat Eag1 K+ channels is conferred by the proximal post-CNBHD region.
BACKGROUND
In mammals, Eag K+ channels (KV10) are exclusively expressed in the brain and comprise two isoforms: Eag1 (KV10.1) and Eag2 (KV10.2). Despite their wide presence in various regions of the brain, the functional role of Eag K+ channels remains obscure. Here we address this question by characterizing the subcellular localization of rat Eag1 (rEag1) and rat Eag2 (rEag2) in hippocampal neurons, as well as determining the structural basis underlying their different localization patterns.


RESULTS
Immunofluorescence analysis of young and mature hippocampal neurons in culture revealed that endogenous rEag1 and rEag2 K+ channels were present in both the dendrosomatic and the axonal compartments. Only rEag1 channels displayed a punctate immunostaining pattern and showed significant co-localization with PSD-95. Subcellular fractionation analysis further demonstrated a distinct enrichment of rEag1 in the synaptosomal fraction. Over-expression of recombinant GFP-tagged Eag constructs in hippocampal neurons also showed a significant punctate localization of rEag1 channels. To identify the protein region dictating the Eag channel subcellular localization pattern, we generated a variety of different chimeric constructs between rEag1 and rEag2. Quantitative studies of neurons over-expressing these GFP-tagged chimeras indicated that punctate localization was conferred by a segment (A723-R807) within the proximal post-cyclic nucleotide-binding homology domain (post-CNBHD) region in the rEag1 carboxyl terminus.


CONCLUSIONS
Our findings suggest that Eag1 and Eag2 K+ channels may modulate membrane excitability in both the dendrosomatic and the axonal compartments and that Eag1 may additionally regulate neurotransmitter release and postsynaptic signaling. Furthermore, we present the first evidence showing that the proximal post-CNBHD region seems to govern the Eag K+ channel subcellular localization pattern.
Chao-Chin
CC
Chuang
Guey-Mei
GM
Jow
Huei-Min
HM
Lin
Yu-Han
YH
Weng
Jui-Hsiang
JH
Hu
Yi-Jheng
YJ
Peng
Yi-Chih
YC
Chiu
Mei-Miao
MM
Chiu
Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, No, 155, Section 2, Li-Non Street, Taipei 12212, Taiwan. cjjeng@ym.edu.tw.
Chung-Jiuan
CJ
Jeng
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D006624:Hippocampus / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions; D014018:Tissue Distribution
D051638:Ether-A-Go-Go Potassium Channels; C496886:Kcnh1 protein, rat; C402401:Kcnh5 protein, rat