Abstract
The cardiac L-type calcium channel is a multi-subunit complex that requires co-assembling of the pore-forming subunit CaV1.2 with auxiliary subunits CaVα2δ and CaVβ. Its traffic has been shown to be controlled by these subunits and by the activation of various G-protein coupled receptors (GPCR). Here, we explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Bioluminescence Resonance Energy Transfer (BRET) assay between β-arrestin and L-type channels in angiotensin II-stimulated cells was used to assess the functional consequence of AT1R activation, while immunofluorescence of adult rat cardiomyocytes revealed the effects of GPCR activation on CaV1.2 trafficking. Angiotensin II exposure results in β-arrestin1 recruitment to the channel complex and an apparent loss of CaV1.2 immunostaining at the T-tubules. Accordingly, angiotensin II stimulation causes a decrease in L-type current, Ca2+ transients and myocyte contractility, together with a faster repolarization phase of action potentials. Our results demonstrate that prolonged AT1R activation induces β-arrestin1 recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling.
Highlights
We explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking
Given the fact that Bioluminescence Resonance Energy Transfer (BRET) can only occur between particles located within ~100 Å from each other, this approach enables the study of the angiotensin II (AngII)-mediated interaction between β-arrestin and CaV1.2 channels in live cells
To test if the AT1 receptor (AT1R) activation has functional consequences on CaV1.2, we explored the effect of AngII on endogenous L-type calcium currents in adult rat cardiomyocytes
Summary
We explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Our results demonstrate that prolonged AT1R activation induces β-arrestin[1] recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling. Activated AT1R is phosphorylated by GPCR kinases (GRKs) and subsequently β-arrestin is recruited to the receptor This receptor/β-arrestin interaction leads to receptor desensitization, ending the G protein-dependent signaling by interfering with the coupling between the G protein and its receptor. Most of these interactions lead to endocytosis of the channel following activation of the respective receptor[8, 9, 11, 13]
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