A naturally occurring truncated Cav1.2 α1-subunit inhibits Ca2+current in A7r5 cells
| Autor: | Samantha J. Fromme, Robert H. Cox |
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| Rok vydání: | 2013 |
| Předmět: |
Male
Patch-Clamp Techniques Calcium Channels L-Type Physiology Myocytes Smooth Muscle Transfection Cav1.2 Cell Line Exon Animals Humans Patch clamp Base Sequence biology Endoplasmic reticulum Alternative splicing HEK 293 cells Articles Cell Biology Molecular biology Protein Structure Tertiary Rats Alternative Splicing HEK293 Cells Cell culture biology.protein |
| Zdroj: | American Journal of Physiology-Cell Physiology. 305:C896-C905 |
| ISSN: | 1522-1563 0363-6143 |
| DOI: | 10.1152/ajpcell.00217.2013 |
| Popis: | Alternative splicing of the voltage-gated Ca2+(CaV) α1-subunit adds to the functional diversity of Ca2+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 CaVα1-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 β2and α2δ1accessory 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 Ca2+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 Ca2+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. |
| Databáze: | OpenAIRE |
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