Dataset Information

Translocatable voltage-gated Ca2+ channel β subunits in α1-β complexes reveal competitive replacement yet no spontaneous dissociation.

ABSTRACT: β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

SUBMITTER: Yeon JH

PROVIDER: S-EPMC6196550 | biostudies-other | 2018 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Translocatable voltage-gated Ca2+ channel β subunits in α1-β complexes reveal competitive replacement yet no spontaneous dissociation.

Yeon Jun-Hee JH Park Cheon-Gyu CG Hille Bertil B Suh Byung-Chang BC

Proceedings of the National Academy of Sciences of the United States of America 20180926 42

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without ...[more]

PMID: 30257950

Similar Datasets

Project description:Background:Voltage-gated sodium (NaV) channels are heteromeric proteins consisting of a single pore forming ?-subunit associated with one or two auxiliary ?-subunits. These channels are classically known for being responsible of action potential generation and propagation in excitable cells; but lately they have been reported as widely expressed and regulated in several human cancer types. We have previously demonstrated the overexpression of NaV1.6 channel in cervical cancer (CeCa) biopsies and primary cultures, and its contribution to cell migration and invasiveness. Here, we investigated the expression of NaV channels ?-subunits (NaV?s) in the CeCa cell lines HeLa, SiHa and CaSki, and determined their contribution to cell proliferation, migration and invasiveness. Methods:We assessed the expression of NaV?s in CeCa cell lines by performing RT-PCR and western blotting experiments. We also evaluated CeCa cell lines proliferation, migration, and invasion by in vitro assays, both in basal conditions and after inducing changes in NaV?s levels by transfecting specific cDNAs or siRNAs. The potential role of NaV?s in modulating the expression of NaV ?-subunits in the plasma membrane of CeCa cells was examined by the patch-clamp whole-cell technique. Furthermore, we investigated the role of NaV?1 on cell cycle in SiHa cells by flow cytometry. Results:We found that the four NaV?s are expressed in the three CeCa cell lines, even in the absence of functional NaV ?-subunit expression in the plasma membrane. Functional in vitro assays showed differential roles for NaV?1 and NaV?4, the latter as a cell invasiveness repressor and the former as a migration abolisher in CeCa cells. In silico analysis of NaV?4 expression in cervical tissues corroborated the downregulation of this protein expression in CeCa vs normal cervix, supporting the evidence of NaV?4's role as a cell invasiveness repressor. Conclusions:Our results contribute to the recent conception about NaV?s as multifunctional proteins involved in cell processes like ion channel regulation, cell adhesion and motility, and even in metastatic cell behaviors. These non-canonical functions of NaV?s are independent of the presence of functional NaV ?-subunits in the plasma membrane and might represent a new therapeutic target for the treatment of cervical cancer.

Dataset Information

Translocatable voltage-gated Ca2+ channel β subunits in α1-β complexes reveal competitive replacement yet no spontaneous dissociation.

Publications

Translocatable voltage-gated Ca<sup>2+</sup> channel β subunits in α1-β complexes reveal competitive replacement yet no spontaneous dissociation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets