C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits

• Published in: The Journal of biological chemistry 2001-06, Vol.276 (24), p.21089
• Main Authors: Gao, T, Cuadra, A E, Ma, H, Bunemann, M, Gerhardstein, B L, Cheng, T, Eick, R T, Hosey, M M
• Format: Article
• Language: English
• Subjects: Animals; Barium - pharmacology; Calcium Channels, L-Type - chemistry; Calcium Channels, L-Type - drug effects; Calcium Channels, L-Type - physiology; Cell Line; Cell Membrane - physiology; Humans; Kinetics; Mammals; Membrane Potentials - drug effects; Membrane Potentials - physiology; Patch-Clamp Techniques; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Protein Subunits; Recombinant Proteins - chemistry; Recombinant Proteins - drug effects; Recombinant Proteins - metabolism; Sequence Deletion; Transfection
• ISSN: 0021-9258

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.