CaV1.2 Interaction with AT1R Reduces Receptor Internalization

Expression of modified CaV1.1 constructs in dysgenic (CaV1.1 null) myotubes has been useful in the determination of regions of the CaV1.1 that are important for skeletal muscle excitation-contraction (EC) coupling, however the use of this system to probe CaV1.1 channel gating has been problematic because dysgenic myotubes express a variety of other ion channels which conduct Ca2þ and contribute to intramembrane gating charge movement. This obstacle can be overcome by recording from tsA201 cells coexpressing CaV1.1 a1S, b1a, a2d1 and Stac3 subunits. Our group has previously characterized, in dysgenic myotubes, a CaV1.1 mutation (R174W) which affects the innermost basic residue of the voltage-sensing S4 a-helix of Repeat I; R174W abolished activation of the L-type Ca2þ current in response to depolarization without affecting the magnitude or voltage-dependence of charge movement. Using tsA201 cells, we show that this dysfunction arises from an impaired gating transition of the RI voltage-sensor that is not detectable with short (20 ms) depolarizations. Specifically, we were unable to recruit the documented slow-moving gating charge component attributable to RI with longer (200 ms) test potentials. We next assessed the effects of corresponding mutations in Repeats II (K537W), III (R906W) and IV (K1245W) in order to determine the way in which translocation of these voltage-sensors affects channel opening. Currents and intramembrane charge movements produced by R906W were both smaller and shifted (~10 mV) to more depolarizing test potentials. In contrast to the severe loss of function observed with R174W and the somewhat impaired gating of R906W, the K537W and K1245W mutants produced L-type currents with enormous peak amplitudes almost three times greater than wild-type CaV1.1 and with greatly impaired deactivation. Supported by Conacyt 169006 (UM), the Boettcher Foundation (RAB), and NIH AR055104, NIH AR052354 and MDA 277475 (KGB).