Calcium entry into excitable cells can be regulated by controlling both the activity of calcium channels and the amount of channels available at the plasma membrane. Little is known about their internalisation and degradation. One post-translational modification shown to be involved in membrane protein internalisation and subsequent degradation is the attachment of ubiquitin moieties by ubiquitin-ligases. Previously, it has been shown that cardiac ion channels such as Nav1.5 and KCNQ1 are down-regulated by ubiquitin-ligases of the Nedd4 family. These regulations involve the interaction between the PY motif of the target channels and the WW motif of the ubiquitin-ligases. Despite the absence of such PY motif in the cardiac voltage-gated calcium channel Cav1.2, we investigated whether this channel could by regulated in the same manner than other cardiac channels as previously described.
We co-expressed, in HEK293 cells, L-type calcium channels and its two regulatory subunits Cavbeta and Cavalpha2delta1 together with ubiquitin-ligases, and examined by voltage-clamp whole-cell recordings the calcium current. We next determined by western blot and surface biotinylation assays the availability of the different subunits of calcium channels in total HEK293 lysates, and at the cell surface. Levels of ubiquitylation of the different subunits were assessed by pull-down GST-S5A and immunoprecipitation of Cav1.2 and its subunits.
We found that co-expressing the ubiquitin-ligase Nedd4-1 significantly reduced Cav currents, and decreased Cavalpha and its subunit protein levels. This effect was Nedd4-1 specific since none of the other members of the Nedd4 family we tested produced a similar effect. We also found that the effect of Nedd4-1 was dependent on the co-expression of the Cavbeta subunit. No Nedd4-1-dependent increase in ubiquitylation of the Cavalpha protein was found; and unexpectedly, the two regulating subunits Cavbeta and Cavalpha2delta1 were detected to be deubiquitylated upon Nedd4-1 co-expression.
Our data suggest that Nedd4-1 regulates the expression of Cav channels and its subunits by Nedd4-1 via an indirect mechanism constituting a new regulatory pathway to be determined. Further experiments will focus on the role of adrenergic receptors known to be ubiquitylated by Nedd4 and to bind to Cav1.2.Le texte complet de cet article est disponible en PDF.