L-Type Voltage-Gated Ca²⁺ Channel C-Terminal Proximal and Distal Domains (PCRD and DCRD) Bind to the IQ-Motif and May Modulate Channel Function

The L-type voltage-gated Ca 2+ channel (Ca V 1.2) controls gene expression, cardiac contraction, and neuronal activity. The C-terminal cytosolic region of the Ca V 1.2 alpha subunit (α1C) contains two domains known as proximal and distal C-terminal regulatory domains (PCRD and DCRD), which have been suggested to control Ca 2+ -dependent channel inactivation (CDI). Previous studies identified a salt bridge interaction between PCRD and DCRD that might be central to channel function. In this study, we expressed and purified recombinant constructs of PCRD (residues 1680–1750) and DCRD (residues 2035–2089) in E. coli for NMR structural analysis. PCRD and DCRD each exhibit backbone NMR chemical shifts consistent with a random coil and lack of tertiary structure. A disordered random coil structure may explain the absence of electron density for these domains in recent cryo-EM structures of Ca V 1.2. Despite this lack of structure, fluorescence polarization binding assays reveal PCRD and DCRD each bind to the Ca V 1.2 IQ-motif (residues 1644–1668) with dissociation constants of 1.5 ± 1 and 14 ± 5 μM, respectively. Both PCRD and DCRD also become insoluble in the presence of high micromolar levels of the IQ peptide, consistent with each domain forming an insoluble complex with the IQ peptide. AlphaFold3 predicts that DCRD adopts a 3-helix bundle that binds to the helical IQ-motif, while PCRD was previously suggested to form a 4-helix bundle. We propose that the PCRD and DCRD bind to opposite sides of the helical IQ-motif, which may oppose calmodulin (CaM) binding to Ca V 1.2 and thus modulate channel CDI.