Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction
Background—L-type CaV1.2 channels play crucial roles in regulation of blood pressure. Galectin-1 (Gal-1), has been reported to bind to the I-II loop of CaV1.2 channels to reduce their current density. However, the mechanistic understanding for the down-regulation of CaV1.2 channels by Gal-1, and whether Gal-1 plays a direct role in blood pressure regulation remain unclear.
Methods—In vitro experiments involving co-IP, western blot, patch-clamp recordings, immunohistochemistry and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 down-regulates CaV1.2 channel in transfected HEK 293 cells, smooth muscle cells, arteries from Lgasl1-/- mice, rat and human patients. In vivo experiments involving delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting CaV1.2-Gal-1 interaction on blood pressure monitored by tail cuff or telemetry methods.
Results—Our study reveals that Gal-1 is a key regulator for proteasomal degradation of CaV1.2 channels. Gal-1 competed allosterically with CaVβ subunit for binding to the I-II loop of CaV1.2 channel. This competitive disruption of CaVβ binding led to CaV1.2 degradation by exposing the channels to poly-ubiquitination. Notably, we demonstrated that the inverse relationship of reduced Gal-1 and increased CaV1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice due to up-regulated CaV1.2 protein level in arteries. To directly regulate blood pressure by targeting the CaV1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1, by a mini-osmotic pump and this specific disruption of CaV1.2-Gal-1 coupling increased smooth muscle CaV1.2 currents, induced larger arterial contraction and caused hypertension in rats. In contrasting experiments, over-expression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats.
Conclusions—We have defined molecularly that Gal-1 promotes CaV1.2 degradation by replacing CaVβ and thereby exposing specific lysines for poly-ubiquitination, and by masking I-II loop ER export signals. This mechanistic understanding provided the basis for targeting CaV1.2-Gal-1 interaction to demonstrate clearly the modulatory role Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension.
- Received August 21, 2017.
- Revision received March 8, 2018.
- Accepted March 28, 2018.