A naturally occurring truncated Cav1.2 α1-subunit inhibits Ca2+ current in A7r5 cells.
Alternative splicing of the voltage-gated Ca(2+) (CaV) α1-subunit adds to the functional diversity of Ca(2+) channels. A variant with a 73-nt deletion in exon 15 of the Cav1.2 α1-subunit (Cav1.2Δ73) produced by alternative splicing that predicts a truncated protein has been described, but its function, if any, is unknown. We sought to determine if, by analogy to other truncated CaV α1-subunits, Cav1.2Δ73 acts as an inhibitor of wild-type Cav1.2 currents. HEK-293 cells were transfected with Cav1.2Δ73 in a pIRES vector with CD8 or in pcDNA3.1 with a V5/his COOH-terminal tag plus β2 and α2δ1 accessory subunits and pEGFP. Production of Cav1.2Δ73 protein was confirmed by Western blotting and immunofluorescence. Voltage-clamp studies revealed the absence of functional channels in transfected cells. In contrast, cells transfected with full-length Cav1.2 plus accessory subunits and pEGFP exhibited robust Ca(2+) currents. A7r5 cells exhibited endogenous Cav1.2-based currents that were greatly reduced (>80%) without a change in voltage-dependent activation when transfected with Cav1.2Δ73-IRES-CD8 compared with empty vector or pIRES-CD8 controls. Transfection of A7r5 cells with an analogous Cav2.3Δ73-IRES-CD8 had no effect on Ca(2+) currents. Immunofluorescence showed intracellular, but not plasma membrane, localization of Cav1.2Δ73-V5/his, as well as colocalization with an endoplasmic reticulum marker, ER Organelle Lights. Expression of Cav1.2Δ73 α1-subunits in A7r5 cells inhibits endogenous Cav1.2 currents. The fact that this variant arises naturally by alternative splicing raises the possibility that it may represent a physiological mechanism to modulate Cav1.2 functional activity.
Program in Cardiovascular Studies, Lankenau Institute for Medical Research, Main Line Health System, Wynnewood, Pennsylvania.
Robert H
RH
Cox
Samantha J
SJ
Fromme
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D014162:Transfection
C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type
A7r5 cells; Cav1.2 subunits; alternative splicing; endoplasmic reticulum; truncated Cav1.2 transcripts