Expression of GFP-rEag1 and GFP-rEag2 channels in HEK293T cells and hippocampal neurons. (A) Immunofluorescence staining and functional expression of GFP-rEag1 and GFP-rEag2 K+ channels in HEK293T cells. GFP fluorescence (shown in green) and rEag1/rEag2 immunofluorescence (shown in red) signals demonstrated lucid co-localization at the membrane region. Scale bar, 10 μm. Whole-cell patch clamp parameters: the holding potential for rEag1 and rEag2 was -90 and -110 mV, respectively; the pulse protocol comprised 300-ms depolarizing test pulses ranging from -70 to +50 mV (rEag1) or from -90 to +30 mV (rEag2), with 10-mV increments. (B) Over-expression of GFP-rEag1/rEag2 in DIV12 hippocampal neurons. GFP signal is shown in green, and MAP2 immunofluorescence signal in red. Note the presence of prominent GFP puncta for rEag1, but not rEag2. Scale bar, 25 μm. (C) (Top) Schematic representation of the structural topology of Eag K+ channel. (Bottom) Protein sequence alignment between rEag1 and rEag2 over the post-CNBHD region. Yellow shade: identical residues. Green shade: homologous residues. Sequence alignment analysis was implemented with the Vector NTI software (InforMax).