Irreversible Binding of Ca2+ Channel β Subunit to α1B Revealed by Chemically-Inducible Dimerization System

Abstract

Voltage-gated calcium (CaV) channel β subunit increases the expression of pore-forming α1 subunit in the plasma membrane and regulates the biophysical properties of channel gating. In particular, the gating of high-voltage activated (HVA) Ca2+ channels is differentially controlled by CaV β subunits depending on the isotypes and intracellular location. However, the molecular mechanism of type specificity and cellular location of β subunit in CaV channel regulation is not clearly determined. We confirmed the constitutive localization of β2a in the plasma membrane irrelevant of the existence of α1B and α2δ1 is due to palmitoylation of N-terminus, and that CaV2.2 current with β2a is slowly inactivated compare to the cells with other types of cytosolic β subunits. In order to further understand the functional role of palmitoylation in the formation of channel complex and current regulation, we constructs a translocatable β2a(C3,4S)-FG a palmitoylation-resistant form of β2a, which is by tagged the β2a(C3,4S) with a FKBP domain and a green fluorescent protein (GFP) sequentially on its C-terminus as a chemically-inducible dimerization (CID) system. When the mutant β2a(C3,4S)-FG was expressed in tsA201 cells, it was mainly located in the plasma membrane in the presence of α1B, while it distributes throughout the cytosol without the α1B. Since the palmitoylation does not occur in the mutant form, the targeting of β2a(C3,4S)-FG to the plasma membrane can be only due to the interaction between α1B and β subunits. We also found that β2a(C3,4S)-FG and β2b-FG are not translocated to the cytosolic organelles by the application of rapamycin. Thus, the results demonstrate that the interaction between α1B and β2a subunits is irreversible.