a. Schematic diagram of the α1-subunit for neuronal L-type calcium channels. The third domain is highlighted, showing the amino acids’ sequence of the S5 segment that confers 1,4-dihydropyridine (DHP) sensitivity: CaV1.3-T1081 and CaV1.2-T1066. b. T1081 on the CaV1.3 subunit was mutated to Y, which should render the channels insensitive to DHPs. c. Both 5 μM isradipine (blue) and 100 μM cp-PYT (red) failed to significantly inhibit currents in CaV1.3-T1081Y channels, suggesting a common binding site. d. Box plot summarizing the percent inhibition observed after application of either 5 μM isradipine (blue) or 100 μM cp-PYT (red) to wild-type CaV1.3 (isradipine: n = 5, median = 90%; cp-PYT: n = 7, 60%) and CaV1.3-T1081Y (isradipine: n = 5, median = 1%; cp-PYT: n = 5, median = 2%) Ca2+ channels. Both the isradipine and cp-PYT effects were statistically significant (Mann-Whitney Rank Sum Test, **p < 0.001). e. SKP004D11, a potent and CaV1.3 selective inhibitor was tritiated in order to label the cp-PYT binding site on CaV13 L-type calcium channels. The specific binding of 3H2-SKP004D11 was determined with a displacement assay using non-labeled SKP004D11 (n = 6, median specific binding = 59%). f.
3H2-SKP004D11 specific binding was displaced from CaV1.3 LTCCs in dissociated HEK293 membranes in a dose-dependent manner that was fit with a Langmuir isotherm (Ki = ~60 nM; n = 4; means are plotted). g. Scatchard plot of the 3H2-SKP004D11 displacement data from CaV1.3 channels in dissociated membrane preparations (red; mean values from four independent experiments). The Kd of SKP004D11 for CaV1.3 LTCCs was derived from the negative-inverse of the slope from the Scatchard plot (n = 4, median = 40 nM). h. Isradipine displaced 3H2-SKP004D11 (Ki = 68 nM). This was determined by a least-squares fit of a Langmuir isotherm and employment of the Cheng-Prusoff equation. For all binding experiments the 3H2-SKP004D11 concentration was 3.0 nM and membrane protein concentrations for stably expressed CaV1.3 LTCCs in HEK-293 cells were between 0.1 and 0.4 mg/mL (see Experimental Section: Binding Experiments). i. Isradipine binding to HEK293 membranes is dependent on the presence of Cav1.3 channel protein.