Figure 1. Membrane expression of the full-length extracellularly HA-tagged Ca_V1.3_L calcium channel in hippocampal neurons.

(a) Representative hippocampal neuron (21 DIV) transfected with the HA-tagged full-length Ca_V1.3_L and labeled with anti-HA (live staining) and eGFP (to visualize cell morphology). Inset shows the cell soma at reduced contrast to visualize the clustered distribution of Ca_V1.3_L. (b,c) Labeling of Ca_V1.3_L (b) on the dendritic surface showed similarly distributed clusters (examples indicated by arrowheads) as Ca_V1.2 (c) on the shaft and on dendritic spines. Co-transfection of the auxiliary subunit β_4b (V5-tagged, anti-V5) increased total surface expression without changing the localization of Ca_V1.3_L clusters ((b) lower panel). (d) Quantification of calcium channel surface expression. Total surface expression of Ca_V1.3_L (total surface area on the dendritic shaft occupied by calcium channel clusters) is 29% of Ca_V1.2. Co-expression of the β_4b subunit caused a 4-fold increase of Ca_V1.3_L surface expression. Values for individual cells (dots) and the means (line) for each condition are shown. Statistics: ANOVA with Holm-Sidak posthoc analysis (F_2,57 = 16.6; p < 0.001; posthoc: ***p < 0.001). Data from 2 culture preparations and 6 independent experiments, between 15 and 29 cells were analyzed in each condition. Scale bar, 20 μm (a) and 5 μm (b,c).