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Fig. 2. Endogenous expression of KCNQ channel subunits in primary hippocampal neurons. (A) Reverse-transcription PCR analysis from cultured rat hippocampal neurons. RT-PCR was performed using 0.15 µg of DNase I-treated, total RNA from cultured hippocampal neurons (10 DIV). KCNQ2 (Q2), KCNQ3 (Q3), KCNQ4 (Q4) and KCNQ5 (Q5) were detected in the cultures. M, Marker; Q1, KCNQ1. (B) Immunoblot analysis of endogenous expression of KCNQ2 and KCNQ3 in cultured hippocampal neurons. Rat brain membranes (RBM) and lysates of primary hippocampal neurons (HN, 10 DIV) were subjected to western blotting and examined for KCNQ2 and KCNQ3 expression. Stars indicate bands that correspond to KCNQ2 and KCNQ3, respectively. Both channel subunits were detected in the hippocampal cultures. (C) Immunodetection of endogenous KCNQ2 and KCNQ3 in the hippocampal cultures. Hippocampal neurons (21 DIV) were double-labeled for endogenous KCNQ2 and MAP2 or endogenous KCNQ3 and MAP2. Both KCNQ2 and KCNQ3 displayed intracellular, somatic localization as well as localization to the AIS (MAP2-negative). Bar, 20 µm. Arrows mark the position of the AIS, while arrowheads point to the distal axon. (Right) Analysis of the immunofluorescence intensity profiles of endogenous KCNQ2 and endogenous KCNQ3 along the main axon up to 100 µm from the soma from the two images demonstrated that the two subunits were enriched in the AIS compared to the distal axon. (D) Electrophysiological detection of endogenous KCNQ subunits. Shown are currents recorded from a cultured neuron (10 DIV) in the absence (control) and presence of the KCNQ-specific blocker XE-991. Whole-cell currents were recorded from voltage-clamped neurons, and a standard hyperpolarizing protocol (top traces) was used to measure the M-current. Cells were held at –70 mV and then depolarized to –20 mV before they were stepped back to hyperpolarized voltages to measure the M-current. Difference currents were obtained by subtracting the control trace at –40 mV from that in the presence of XE-991. Below is shown a time series of a slowly activating current at –40 mV recorded every 5 seconds in the absence or presence of XE-991. The current is stable in the whole cell recording configuration and is partially blocked by 10 µM XE-991. (E) Immunoblot detection of KCNQ2 expression in KCNQ2-transfected COS-1 cells (COS-1 Q2), rat brain membranes (RBM), primary hippocampal neurons (HN) and KCNQ2-transfected primary hippocampal neurons (HN-Q2). Note the high level of exogenous KCNQ2 expression compared with the level of endogenous KCNQ2.
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