Figure 4. Functional characterization of the novel Best1V1 splice variant missing exon 2 by heterologous expression in HEK293 cells.

A, Western blot analysis of Best1V1 and Best1V1Δex2 heteroexpressed in HEK293 and probed with a monoclonal anti-Best1 antibody. Untransfected HEK293 (WT) cell lysates shown as a control for signal specificity. The Western blots membranes were stripped and reprobed with a monoclonal anti-actin antibody to compare protein loading (shown in box below). The figure is a representative image of three Western blots in independent cell transfections. B, Representative traces of Best1V1Δex2-mediated Cl⁻ currents recorded in response to step pulses from −100 mV to +100 mV in 20 mV increments. C, Representative traces showing the current-voltage relationship of heteroexpressed Best1V1Δex2 channels in the absence and presence of 200 μM niflumic acid (NFA). D, Summary of the electrophysiological recording performed in HEK293 cells expressing Best1V1Δex2. Data are the mean values ±SE of average current densities recorded with [Ca²⁺] in the pipette solution buffered at 0 or 1 μM, in the presence or absence of 200 μM NFA. The recordings were done in two independent cell transfections, with the number of cells indicated above each bar. Not all Best1V1Δex2-trensfectected cells showed Ca²⁺-sensitive Cl⁻ currents (see text for further details). ***p<0.001, vs. currents recorded at [Ca²⁺]_pipette = 0 μM; ^##p<0.01 vs. currents recorded in the absence of NFA.