Fig. 4. Co-expressed hKCNQ2-cmyc and hKCNQ3-FLAG are localized to the AIS of primary hippocampal neurons. Hippocampal neurons were co-transfected with hKCNQ2-cmyc and hKCNQ3-FLAG, fixed and triple-labeled for hKCNQ2-cmyc, hKCNQ3-FLAG and either ankyrin-G (A) or MAP2 (B). Bar, 20 µm. hKCNQ2-cmyc and hKCNQ3-FLAG labeling was enriched in the AIS, which is positive for ankyrin-G, but devoid of MAP2. Additional staining could also be observed intracellularly as well as in the more distal parts of the axon. Arrows mark the position of the AIS, while arrowheads point to the distal axon. (C) Representative current traces of an hKCNQ2-cmyc/hKCNQ3-FLAG transfected neuron in the absence or presence of 10 µM XE-991. Protocols were as described in Fig. 2. A large, slowly non-inactivating M-current could be measured in transfected neurons. For comparison with the endogenous M-current, the bottom trace shows a time-series of a transfected neuron (Q2+Q3) and a GFP-transfected neuron (Control) before and after application of XE-991 (10 µM). The level of XE-991-sensitive current is on average increased 15-fold in neurons transfected with hKCNQ2-cmyc/hKCNQ3-FLAG compared with that in controls (see Fig. 4B for quantification).