Nature 2020 01 30 Part.02

Nature | Vol 577 | 30 January 2020 | 697

on the CaV1.2 proteome signature in Langendorff-perfused whole
hearts (Fig. 2g, h). We detected isoproterenol-induced recruitment
of the PKA catalytic subunit (PKAcat) to CaV1.2 channels in cardiomyo-
cytes isolated from both α1C–APEX2 and β2B–APEX2 mice (Fig. 2e,
f). All three approaches (using isolated cardiomyocytes from α1C–
APEX2 and β2B–APEX2 mice, and α1C–APEX2 whole hearts) indicated
a 30–50% decrease in the amount of the small Ras-like G protein Rad
in the neighbourhood of CaV1.2 following application of isoproter-
enol (Fig. 2e, f, h, Extended Data Figs. 4a–c, 5a, b and Supplementary
Tables 3–5). By analysing the overlap of proteins displaying isopro-
terenol-induced changes in α1C–APEX2 and β2B–APEX2 experiments,
we found Rad to be the only candidate protein displaying this behav-
iour (Extended Data Fig. 5b, c). By contrast, a 10-minute exposure of
cardiomyocytes isolated from nontransgenic mice to isoproterenol
(Fig. 2i) had a minimal effect on proteins relatively quantified by
TMT SPS MS^3 , and specifically no substantial effect on the amount
of Rad by comparison with untreated paired cardiomyocytes (Fig. 2j,
Extended Data Fig. 4d and Supplementary Table 6). In this control
experiment, proteins were not proximity labelled. Thus, β-adrenergic

stimulation depletes Rad from the neighbourhood of CaV1.2, but not

from the cell as a whole.

Rad is a member of the Rad, Rem, Rem2 and Gem/Kir (RGK) family

of Ras-like GTP-binding proteins—known for their capacity to inhibit

all high-voltage-activated Ca2+ channels^8 ,^9 —and is a potential PKA tar-

get^20. Rad-knockout mice display an increased basal Ca2+ current with

activation at lower voltages and reduced β-adrenergic stimulation of

their Ca2+ channels^21 ,^22. Other studies, however, led to expectations that

Rad is not directly involved in adrenergic regulation of CaV1.2, because

overexpression of Rad^23 or Rem^24 in cultured cardiomyocytes markedly

attenuated the basal CaV1.2 current, which was also not increased by

β-adrenergic stimulation. Nevertheless, our proximity labelling results

elevated Rad as the leading candidate for the critical missing link that

enables PKA regulation of CaV1.2.

PKA regulates CaV1.2 via Rad phosphorylation

The robust heterologous reconstitution of PKA regulation of CaV1.2

currents has long been pursued as a crucial step in identifying and

Tryptic digest

TMT labelling

phosphorCheck PLylatioBn

iso

iso

NTG

Time (min) 010

Lysis

cardiomyocytesIsolated

–2 –1 012

1 × 10 –4

0.1

1.0

log 2 -transformed fold change (isoproterenol/no isoproterenol) log 2 -transformed fold change (isoproterenol/no isoproterenol)

log 2 -transformed fold change (isoproterenol/no isoproterenol) log 2 -transformed fold change (isoproterenol/no isoproterenol)

PKAcat

Rad

P = 0.05

P = 0.05

β2B–APEX

–2 –1 012

0.001

0.001

0.01

0.01

0.1

1.0

Pval

ue

Pvalu

e

Pval

ue

Pvalu

e

Rad

PKAcat

–2 –1 012

0.001

0.01

0.1

1.0

Rad

–2 –1 012

1 × 10 –6

1 × 10 –5

1 × 10 –4

0.001

0.01

0.1

1.0 Rad

P = 0.0 5

P = 0.0 5

Biotin

OH

iso

iso

Wholeheart

Monitor ECG

Time (min) 0 5 10 15

400bpm

262bpm 15 20

H^2 O

2

Quench

Lysis

Check PLB

phosphorylation

Biotin

OH

iso

iso

Time (min) 0 10 20 30

H^2

O^2

Quench

Lysis

Check PLB

phosphorylation

streptavidinDenaturing

pulldown

Tryptic digest

TMT labelling

streptavidinDenaturing

pulldown

Tryptic digest

TMT labelling

α1C–APEX or

α1C–APEX α1C–APEX

whole heart

Isolated

cardiomyocytes

DAPI β2B–APEX Quench

i

b d

c

Anti-V5-Alexa 594

Streptavidin-Alexa 488DAPI

Streptavidin-Alexa 488

f

a

j

e h

g

*NTG

kDa

250

130

α1C–APEX

α1C

–APEX

α1C

–APEX(heart)

β2B–APEX

β2B

-APEX

Input InputPull-down

++++

–+–+

++++

–+–+

Pull-down

Biotin-phenol Biotin-phenol

KV1.5

CaM

RyR2

Jph2

β2B

α1C–APEX

α1C

KV1.5

CaM

RyR2

Jph2

β2B–APEXβ2B

α1C

H 2 O 2 H 2 O 2

(^13095)
(^13095)
(^7255)
(^1309513095)
(^13095)
5572
250
130
250
130
28
17
28
17
(^9572)
55
(^9572)
55
kDa
130250
Fig. 2 | Changes in the CaV1.2 subdomain proteome upon β-adrenergic
agonist activation of PKA signalling in the heart. a, Immunofluorescence of
isolated α1C–APEX2 and β2B–APEX2 cardiomyocytes exposed to biotin–phenol
and hydrogen peroxide. Representative of 13 and 8 cardiomyocytes from 2 and
3 mice respectively. Staining is with anti-V5 and Alexa594-conjugated
secondary antibodies and streptavidin-conjugated Alexa488; nuclear
labelling is with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 5 μm.
b, Immunof luorescence of tissue sections of Langendorff-perfused α1C–APEX2
heart. Scale bars, upper panel, 100 μm; lower panel, 5 μm; lower inset, 5 μm.
Representative of ten sections from two mice. c, Immunoblots of biotin-
labelled proteins from cardiomyocytes of α1C–APEX2 and β2B–APEX2 mice.
CaV1.2 subunits, RyR2, Jph2 and CaM are detected, whereas KV1.5 channels are
not detected, in streptavidin pulldown. Blots representative of three
independent experiments. d, Workf low for processing of isolated
cardiomyocytes. H 2 O 2 , hydrogen peroxide; iso, isoproterenol; PLB,
phospholamban. e, Volcano plot of fold change for relative protein
quantification by TMT mass spectrometry of α1C–APEX2 samples. Data are
means from five pairs of biologically independent samples; non-adjusted
unpaired two-tailed t-test. Levels of Rad in the neighbourhood of CaV1.2 (red
dot) are reduced by 50%, and levels of the PK A catalytic subunit (green dot) are
increased by 50%, upon application of isoproterenol. f, As in e, except for
cardiomyocytes from β2B–APEX2 mice. Data are means from three pairs of
biologically independent samples. Levels of Rad are reduced by 30% and of the
PK A catalytic subunit are increased by 68% in the neighbourhood of CaV1.2
following application of isoproterenol. g, Workf low showing protein labelling
and analysis of Langendorff-perfused α1C–APEX2 mouse hearts. Bpm, beats per
minute. h, As in e,  but for proteins from α1C–APEX2 whole-heart samples. Data
are means for five hearts: five without and five with isoproterenol. Levels of
Rad in the neighbourhood of CaV1.2 are reduced by 36% following application of
isoproterenol. i, Workf low for analysis of isolated cardiomyocytes from non-
transgenic (NTG) mice. j, As in e, but for proteins isolated from NTG
cardiomyocytes without biotinylation or pull-down. The asterisk indicates the
single peptide ESFDSQSLINNQSK, mapping to Uniprot D3Z0N2 (Sorbs2), with
its apparent abundance reduced by isoproterenol treatment in non-transgenic
cardiomyocytes to about 27%, probably because of post-translational
modification. Data are means for four pairs of biologically independent
samples. Levels of Rad in whole heart are unchanged by isoproterenol. For
source gel data, see Supplementary Fig. 1.