Increase of CaV3 channel activity induced by HVA β1b-subunit is not mediated by a physical interaction

Fig. 1. The β1b-subunit increases the current density in all three LVA channels. a–c Representative whole-cell currents recorded at -30 mV from HEK-293 cells transfected with each of the CaV3 channels (CaV3.1, CaV3.2 or CaV3.3) alone or together with the β1b-subunit. Patch-clamp experiments were performed by using a HP of − 100 mV and 5 mM CaCl2 as charge carrier. d–f Current–voltage (I–V) relationships for the same channels as in a–c. For I–V plots data were obtained from currents evocated from − 80 mV to + 80 mV in 10 mV steps; current amplitudes were normalized by cell capacitance to obtain current density values. Smooth lines are fits to data with a modified Boltzmann function (see Experimental Procedures). The corresponding parameters are shown in Additional file 1. g–i Current density (mean ± SEM) at − 30 mV calculated for HEK-293 cells transfected with the indicated CaV3 channels alone or together with β1a, β1b, β2a, β3 or β4 subunits. Only β1b increases current density significantly when transfected with any of the CaV3 channels. Data were normalized to the current density values obtained when CaV3 channels were transfected alone. *Statistical significance when using ANOVA…

Fig. 2. The β1b-subunit increases the amount of CaV channels at the plasma membrane. a Co-localization analysis of HEK-293 cells expressing the α1-subunit of CaV1.2, CaV3.1, CaV3.2 and CaV3.3 fused to GFP, in the absence (-β1b) and presence (+β1b) of the β1b-subunit. Representative confocal microscopy images of cells expressing the respective CaV shown in green (left panels) and the plasma membrane marker FM4-64 (shown in red, middle panel). The co-localization between the CaV α1-subunit and the FM4-64 (in yellow) is shown in Merge panels. The plots to the right show the total pixels of colocalization between the green channel (CaV) and the red channel (FM-464), with the corresponding Pearson’s correlation coefficient (R) for each experimental condition. b The FRET between the GFP in each CaV channel and the blue fluorescent protein in the β1b-subunit. Notice that FRET is observed only between CaV1.2 (HVA channel) and β1b, but not with the other 3 CaV channels (CaV3.1, CaV3.2 and CaV3.3). High and low FRET was calculated pixel-by-pixel and the image shows in pseudo color FRET intensities. The number of cells analyzed for both colocalization and FRET panels were as follows: CaV1.2, 65;…

Fig. 3. CaV3.3 channels and β1b-subunit do not coimmunoprecipitate. Western blot of IP of CaV1.2-GFP with β1b-HA (positive control, lane 1) and CaV3.3-GFP with β1b-HA (lanes 2 and 3). IPs in lanes 1 and 2 were washed three times with lysis buffer after overnight incubation with the indicated proteins, whereas the IP from lane 3 was washed only once. The immunoblot was probed with an antibody against GFP (upper panel), then striped and probed with an anti-HA antibody (middle panel), then striped again and probed with a homemade anti-actin antibody (lower panel). Lanes 4-6 were loaded with 15 µg of total protein from lysates of HEK-293 cells transfected with β1b-HA, CaV1.2-GFP and CaV3.3-GFP, respectively. Finally, lanes 7 and 8 were loaded with 10 µl of the IP supernatant from lanes 1 and 2, accordingly. Notice the importance of exhaustive washing procedures, since incomplete washing of the beads could lead to false positives results. As can be observed, CaV1.2 channels coimmunoprecipitate with β1b-HA (lane 1, upper panel), whereas CaV3.3 channels do not (lane 2, upper panel). Representative figures of three independent experiments