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.
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.
Department of Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
T
T
Gao
A E
AE
Cuadra
H
H
Ma
M
M
Bunemann
B L
BL
Gerhardstein
T
T
Cheng
R T
RT
Eick
M M
MM
Hosey
D000818:Animals; D001464:Barium / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D001464:Barium