RESUMEN
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 ß2 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.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Miocitos del Músculo Liso/metabolismo , Empalme Alternativo , Animales , Secuencia de Bases , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Línea Celular , Células HEK293 , Humanos , Masculino , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Ratas , TransfecciónRESUMEN
BACKGROUND: We have previously demonstrated differences in the gene expression of voltage-gated K v1.X channel alpha-subunits in arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs). The purpose of this study was to test the hypothesis that these differences are also present at the protein level. METHODS: Proteins were isolated from the aorta, mesenteric (MAs) and tail arteries (TAs) of 12- to 15-week-old male WKY and SHR, and analyzed by immunoblotting. K(v) currents were recorded from MA myocytes by patch clamp methods. RESULTS: Expression of Kv1.2, Kv1.5, and Kv2.1 was higher in MAs but was not different in aortas of SHRs as compared to WKYs. In the TA, expression of Kv1.2 and Kv1.5 was higher while that of Kv2.1 was lower in SHR compared to WKY. In the MA, the larger expression of an 80 kDa species of Kv1.2 in SHRs was associated with a lower expression of a 60 kDa species. Kv2.1 gene expression was larger in MAs from SHRs but not different in TAs. K(v) currents associated with Kv1.X and Kv2.1 channels were both larger in MA myocytes from SHRs but less than expected based upon differences in K(v) alpha-subunit protein expression. CONCLUSIONS: For the MA, K(v) protein expression and current components between WKYs and SHRs were qualitatively consistent, but differences in gene and protein expression were not closely correlated. The higher expression of K(v) subunits in small mesenteric arteries (SMAs) of SHR would tend to maintain normal myogenic activity and vasoconstrictor reserve, and could be viewed as a form of homeostatic remodeling.