A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic–initiated reversal of the small RGK G protein Rad–mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad’s polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad’s N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad’s association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad’s C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.


Determining Membrane Localization with FRET
To monitor changes in Rad localization, we used FRET measurements between a plasma membrane-localized Cerulean fluorophore (the donor) and Venus-tagged Rad (the acceptor).Fluorescence measurements were obtained with a BD Biosciences LSRII flow cytometer equipped with appropriate lasers to excite the donor and the acceptor and bandpass filters to separate the fluorescence emissions.Three fluorescence measurements were obtained from single cells: (1) donor fluorescence emission (excitation: 405 nm and emission/bandwidth: 450/50 nm) denoted as  !, (2) acceptor fluorescence emission due to direct excitation (excitation: 488 nm and emission/bandwidth: 530/30 nm) denoted as  " (), and (3) acceptor fluorescence emission due to FRET (excitation 405 nm and emission/bandwidth: 525/50 nm) denoted as  " ().Each cell is assumed to contain ND donor molecules and NA acceptor molecules.Donor and acceptor do not bind; proximity that supports FRET occurs by random motion.The donor is modified to be attached to the cytoplasmic surface of the plasmalemma and is thus confined to diffuse in two dimensions.A fraction of the acceptors (fmem) is localized to the plasmalemma and the remaining fraction is assumed to diffuse in three dimensions.Significant FRET occurs only when the acceptor is close to the membrane-tethered donor.
FRET efficiency (ED) is obtained from the three fluorescence measurements as described previously (Erickson et al 2003).Briefly, the fluorescence outputs are: Eq. 1 where ND and NA are the numbers of molecules of donor and acceptor, ID and IA are the light intensities of the 405 nm and 488 nm lasers, respectively; GD(λex,D) and GA(λex,A) are instrument-specific constants that incorporate the optical properties of the instrument and the extinction coefficients of the donor and the acceptor, respectively;  !4λ #&,! 6 and  " 4λ #&," 6 are the outputs of the detector in response to the emissions of an excited donor and an excited acceptor.
ED is dependent on the distance (r) between the donor and the acceptor by the Förster relation: where R0 is the Förster distance.R0, in turn, depends on the spectral overlap of the fluorophores, the quantum yield of the donor, and the relative orientation of the fluorophores.This analysis assumes no specific binding of donor and acceptor; rather their distance and relative orientation are determined by random motion.Also, this relation assumes that at most one acceptor is near the donor.Thus, if U(r) denotes the probability density and U(r)dr the probability that for a given donor, there is an acceptor within a distance between r and r + dr, then the expected value of FRET efficiency is given by: Eq. 3 As acceptors can be localized to either the cytoplasm or restricted to the plasmalemma, () =  456 () +  787 () where Ucyt(r) and Umem(r) are probability density functions for acceptors in the cytoplasm and the plasmalemma, respectively.
Eq. 4 Here, for r larger R0, E rapidly converges to 0. As such, the upper limit of the definite integral can be regarded as ∞.Let  " be the total number of molecules of acceptors, and fmem is the fraction of acceptors associated with the membrane.NA*(1-fmem) is the total number of molecules in the cytoplasm of a cell with volume Vcell.The probability of finding an acceptor within a hemispherical shell of radius r and thickness dr centered on the donor is given by:  456 () •  = Similarly, if  " * fmem acceptors are localized to the plasmalemma with surface area SCell, then the probability of finding an acceptor within the annulus of radius r and width dr is: We assume a low concentration of acceptors, such that there is only one acceptor near the donor.Thus, the measured FRET efficiency can be determined by substituting Eqs. 2, 5, and 6 into Eq.4.
If the cell is spherical with radius a, then  *#DD = E F  F and  *#DD = 4 A .Eq. 7 simplifies to: Eq. 8 For Cerulean-Venus FRET pair, R0 ~ 5.2 nm.The average radius of a HEK293 cell, a ~ 6.5 µm.Thus, R0 / a < 0.001 For a sufficiently large fmem, this relationship simplifies to: Eq. 9 From Eq. 1,  " =  ",'()#*+ /( " •  " 4λ #$," 6 •  " 4λ #&," 6).Thus, •  ",'()#*+ Eq. 9 Therefore, if ED is plotted as a function of SA,direct, then the initial slope (i.e.low acceptor concentration) is directly proportional to the fraction of acceptors localized to the plasmalemma.At high acceptor concentrations, it is possible that multiple acceptors are present near the donor.As result, Eq. 2 would require additional terms to account for possible FRET transfer with multiple acceptors, ultimately leading to nonlinearities in the ED-SA,direct relationship.

Supplemental Figure 3 .
Creation of N-2SA knock-in mouse line.(A) Schematic depicting approach for the creation of N-2SA knock-in mouse line.Guides and single-strand oligodeoxynucleotide as the donor template with Ala-substitutions for the two Ser residues were designed by Genome Engineering and iPSC Center (GEiC) at the Washington University in St. Louis.HA-L= homology arm-left, HA-R= homology arm-right.(B) Anti-α1C, anti-Rad and anti-β-actin immunoblots of protein homogenates from WT and N-2SA knock-in mice cardiomyocytes.N=3 mice for each genotype.Supplemental Figure 4. Change in membrane association of Rad induced by altering electrostatics in its C-terminus.(A) Graph of basic hydrophobic (BH) score for the full sequence of Rad, created using a basic-hydrophobic scoring algorithm (36, 41).The x-axis is Rad residue numbers.The y-axis is BH score.BH motifs are defined by peaks with a BH signal > 0.6 (green line).On the right, a Pymol-generated model of C-terminus of Rad.Positive-charged amino acid residues are blue; negative charged amino acid residues are red.(B-C) ED is plotted against SA,direct of Ven-WT Rad, either untreated or treated with 50 μM forskolin + 100 nM calyculin A. (D-E) As in B-C, with C-2SA Rad.(F-G) ED is plotted against SA,direct of Ven-C-2SD Rad and Ven-C-4SD Rad in the absence of forskolin and calyculin A. Supplemental Figure 5. Insertion of negatively charged Asp residues in C-terminus of Rad reduces Rad-mediated inhibition of heterologously expressed Ca 2+ channels.(A) Ba 2+ current elicited by voltage ramp every 6 seconds.Tail current is marked by arrows.(B) Graph of tail current from HEK293 cells heterologously expressed α1C and β2B, and no Rad, WT Rad or Rad with 2, 4, or 6 Asp-residues in C-terminus.Mean ± SEM. ****P<0.0001 by one-way ANOVA, *P<0.05,**P<0.01compared to WT Rad by Dunnett's multiple comparison test.

Figure S1 .
Figure S1.Schematics show a cerulean fluorophore localized to the membrane and relevant geometric arrangement for determining the probability U(r) of finding a Venus fluorophore within a distance r of Cerulean.Left, if Venus is cytosolically localized, it is assumed to diffuse in three dimensions.So, we consider the concentric hemi-spherical shells and the probability of a Venus being within this shell.Right, if Venus is membrane restricted then we assume two-dimensional diffusion.Thus, we consider thin cylindrical shells around Cerulean.

Supplemental Table 1. Parameters of Boltzmann equation fit for I-V curves of full-length CaV1.2.
The table shows mean and SEM values of fits.N=10 oocytes in each group.The statistical analysis was done on raw data (i.e. the values of each parameter in individual oocytes).The asterisks indicate statistically significant differences between parameters in each group vs. the control group (CaV1.2alone).* P <0.05; **** P <0.0001; ns, not significant.Data for all parameters except Gmax were normally distributed (Shapiro-Wilk test).For Gmax, we performed Kruskal-Wallis test followed by Dunn's multiple comparison test.For the other parameters, one-way ANOVA was applied, followed by Tukey pairwise comparison test.