Reconstitution of β-adrenergic regulation of Ca _V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Abstract

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through CaV1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Mapping molecular determinants of CaV2.2 inhibition by RGK proteins and homologs in Xenopus oocytes

“…Recent studies have now shown bona fide physiological relevance for Rad as being a key player in the potentiation of CaV1.2 channel currents during b-adrenergic regulation of cardiomyocytes and HEK cells, as mediated by cAMP and protein kinase A (Ahern et al, 2019;Liu et al, 2020). Our recent work extended this finding, where we showed involvement of Rad in both b1-or b2-mediated adrenergic regulation of CaV1.2 in Xenopus oocytes (Katz et al, 2021). Like Rad, other RGK proteins may play similar physiological role(s) in b-adrenergic regulation of other tissues.…”

“…No significant differences (P=0.138) in IBa were observed between full length CaVβ2b, CaVβ2b-core and CaVβ2bcore with the GS-linker on its C-terminus (Fig. S1 and (Katz et al, 2021)). Therefore, all CaVβcore-Gem chimeras were analyzed in comparison with the CaVβ functional core-GS linker construct.…”

“…The copyright holder for this preprint this version posted June 24, 2022. ; https://doi.org/10.1101/2022.06.21.496996 doi: bioRxiv preprint phosphorylation (Katz et al, 2021;Liu et al 2020), unlike C1-fused RGK proteins whose phorbol ester-dependent membrane association is practically speaking irreversible.…”

“…The copyright holder for this preprint this version posted June 24, 2022. ; https://doi.org/10.1101/2022.06.21.496996 doi: bioRxiv preprint 2012; Yang et al, 2012). Recent studies demonstrate that calcium channel inhibition by RGK proteins is physiologically relevant as Rad plays a central role in adrenergic regulation of CaV1 channels in cardiac myocytes (Ahern et al, 2019;Liu et al, 2020;Katz et al, 2021).…”

Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy

“…To do so, we co-expressed hGluN1a with different mixtures of hGluN2B- wt and hGluN2B-G689C or -G689S mRNAs in Xenopus oocytes (i.e. mRNA titrations [ Berlin et al, 2020 ; Berlin et al, 2011 ; Yakubovich et al, 2015 ; Peleg et al, 2002 ; Katz et al, 2021 ]) and assessed glutamate dose-responses (see Materials and methods). When large mRNA amounts of hGluN2B- wt were co-expressed with very low amounts of hGluN2B-G689C (mRNA ratio: ~16:1, denoted wildtype-high; wt H ), we recorded large currents that readily responded to glutamate ( Figure 4a,b ; wt H ).…”