Rôle de l’extrémité C-terminale dans l’expression du canal calcique Cav1.2 à la membrane plasmique
The L-type calcium channel, Cav1.2, plays an important role in the excitation-contraction coupling of the ventricular myocytes. It has been shown that the alternative splicing of Cavα1.2 subunit could lead to a truncated protein in the C-terminus at exon 45 (Liao, Yong et al. 2005). Many groups have studied deletions in the C-terminus (De Jongh, Warner et al. 1991; Gao, Cuadra et al. 2001). The currents, measured in the whole cell configuration, were significantly higher with the full-length channel. We chose to test some of these deletions (ΔC2030, ΔC1935, ΔC1856, ΔC1733, ΔC1700) in the presence or absence of the Cavβ3 auxiliary subunit which is likely to interact with the C-terminus of the Cavα1.2 subunit through its SH3 domain (Lao, Kobrinsky et al. 2008). The truncated Cavα1.2 subunit, expressed in Xenopus Oocytes, showed macroscopic currents that were greater than those of the full length channel in presence of the Cavβ3 subunit. In addition, the truncated Cavα1.2 subunits displayed currents in the absence of the Cavβ3 subunit in contrast with the Cavα1.2 full length subunit. To investigate whether the larger macroscopic currents resulted in an increase in the number of Cavα1.2 subunits at the plasma membrane, we chose the FACS (« Fluorescence Activated Cell Sorting ») method. An HA-tag was inserted in an extracellular region of the Cavα1.2 subunit and a FITC (« Fluorescein IsoThioCyanate ») coupled anti-HA antibody was used to measure fluorescence. Our results showed that the Cavα1.2-HA subunit of L- type channel is expressed at the plasma membrane in the presence of the Cavβ3 subunit whereas the Cavα1.2-HA subunit is slightly or not expressed at the plasma membrane in its absence. The same results were obtained for the three C-terminal deletions tested under the same conditions (CaVα1.2-HA ΔC1935, CaVα1.2-HA ΔC1856 and CaVα1.2-HA ΔC1733). Taken together, these results suggest that the increased macroscopic currents observed after a partial deletion of the C-terminus is not caused by an increased number of Cavα1.2 proteins expressed at the plasma membrane. Keywords:
Calcium Gating FACS Electrophysiologie Calcium Ion channel Gating Directed Mutagenesis FACS Electrophysiology Western-blot