Cav1.2 Interaction with At1r Reduces Receptor Internalization

The cardiac L type calcium channel is a multi-subunit complex that includes CaV1.2 as the pore-forming subunit that co-assembles with the auxiliary CaVa2d1 subunit and with the CaVb subunit. The main role of these auxiliary subunits is to modify the amount of channels inserted in the plasma membrane. Similarly, trafficking of these channels is controlled by direct interaction with other proteins such as some G-protein coupled receptors (GPCR). Angiotensin II (AngII) type I receptor (AT1R) is a GPCR well known to be involved in the intracellular calcium regulation at the heart, modulating cardiomyocytes contractibility. In this work we show, by co-immunoprecipitation, that AT1R forms a macro-complex with CaV1.2 in primary culture of newborn rat cardiomyocytes and in an heterologous system. Standard biochemical approaches were used to demonstrate that these proteins colocalize at the plasma membrane and to determine the region of CaV1.2 responsible for this interaction. The functional impact of this complex was studied in terms of L-type currents inhibition and in terms of internalization of AT1R. Although inhibition of endogenous L-type current in cardiomyocytes was found to be dependent on the CaVb2 transcriptional start site (TSS) expressed, the presence of the macro-complex was found to be independent on the variant expressed. The effect of this interaction over AT1R internalization was studied in cells stimulated with AngII at different times. As expected, in cell expressing only the AT1R and stimulated with AngII, the receptor is readily internalized after 15 min, however, in cells expressing also CaV1.2, AT1R internalization (and CaV1.2) is observed only after 30 minutes of AngII stimulation. Overall, our results demonstrate that AT1R interaction with CaV1.2 increases the residence time at the plasma membrane of the receptor and induce L-type channel internalization after prolonged AngII exposure.