Fig. 1

Cav3.1 channels exhibit a calcium-dependent association with CaM. Biochemical analysis of binding interactions between Cav3.1 and CaM using the indicated antibodies to immunoprecipitate (IP) or immunoblot (IB). The experiments are representative of at least 3 repetitions. Input lanes reflect controls to verify the efficiency of the IP step should IB antibodies fail to reveal co-IPs. a, b Cav3.1 channels co-IP with CaM from rat brain lysate (a) and homogenates of tsA-201 cells coexpressing Cav3.1 and CaM (b). Co-IPs were conducted in the presence of 100 nM calcium. c The Cav3.1-CaM co-IP is lost for a construct lacking the Cav3.1 C-terminus (Cav3.1ΔCT). Co-IPs were conducted in the presence of 100 nM calcium. d A GST-pull down experiment between the C-terminus of Cav3.1 (CT-Cav3.1) onto CaM beads. GST-CT-Cav3.1 was grown in bacteria, and the GST tag subsequently cleaved off as the GST non specifically bound to CaM beads. CaM beads were instead incubated with purified recombinant Cav3.1 C-terminal peptides in the presence of 100 nM calcium and the sample eluted and run on a Western blot. The blot was probed with an antibody targeting the Cav3.1 C-terminus. e, f Co-IP tests conducted in the indicated calcium concentrations reveal that Cav3.1 associates with CaM only below 5 μM calcium. Co-IPs were conducted from rat brain lysates (e) or from homogenates of tsA-201 cell expressing HA-Cav3.1 and CaM (f). In (f) an HA antibody was used to immunoblot the HA-Cav3.1 conjugate. g Co-IPs from lysates of tsA-201 cells coexpressing Cav3.1 and the CaM EF-hand mutant CaM1234 reveals that CaM1234 and Cav3.1 interact in the presence of 0–1 mM calcium