The punctate localization of rat Eag1 K+channels is conferred by the proximal post-CNBHD region

Abstract

BackgroundIn 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.ResultsImmunofluorescence 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.ConclusionsOur 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.

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

“…We have previously demonstrated that rEag1 channels are significantly expressed at synapses, wherein rEag1 immunofluorescence signals display a distinct punctate localization pattern1012. Similar punctate patterns were also reported by a different research group using single-particle-tracking techniques to characterize the presynaptic localization of rEag1 channels13.…”

“…In Drosophila , where Eag1 K + channels were initially identified, mutations in the eag gene result in distinct defects including hyper-excitability of neuromuscular junction and failure in learning courtship conditioning behavior1415, suggesting an important role of Eag1 channels in synaptic transmissions. Consistent with this notion, mammalian Eag1, but not Eag2, is significantly expressed within the synaptic region and displays a distinct punctate localization pattern101213.…”

“…Previous biochemical and immunofluorescence studies from our lab strongly suggest that rEag1 is present at both presynaptic and postsynatipc regions in neurons1012. We therefore asked whether Cul7 and rEag1 shared overlapping subcellular localization in neurons by performing fractionation analyses, which separate adult rat forebrain homogenates into multiple subcellular fractions.…”

“…In mammals, there are two Eag isoforms: Eag1 (K V 10.1) and Eag2 (K V 10.2); both isoforms are widely expressed in many areas of the brain and exhibit differential subcellular localization in axonal and dendrosomatic compartments2345678910111213. In Drosophila , where Eag1 K + channels were initially identified, mutations in the eag gene result in distinct defects including hyper-excitability of neuromuscular junction and failure in learning courtship conditioning behavior1415, suggesting an important role of Eag1 channels in synaptic transmissions.…”

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

“…For biochemical characterization, Myc-tagged rEag1 (Myc-rEag1) was co-expressed with either GFP-tagged rEag1 (GFP-rEag1) or FLAG-tagged hErg (FLAG-hErg) in HEK293T cells for the co-immunoprecipitation experiment. Despite the large size of GFP, previous observations from our laboratory indicated that adding the GFP tag fails to significantly affect the assembly and trafficking of Eag subunits (22). Fig.…”