Nature 2020 01 30 Part.02

Nature | Vol 577 | 30 January 2020 | 699

We used a flow-cytometry Förster resonance energy transfer (FRET)

two-hybrid assay^30 to probe potential PKA-mediated changes in the

binding of β2B subunits and wild-type Rad. At baseline, we detected

robust binding between cerulean-tagged β2B subunit and venus-tagged

wild-type Rad, consistent with previous studies^31 (with an effective

dissociation constant (Kd,EFF) of 7,957 ± 418; Fig. 4b and Extended Data

Fig. 9c). Coexpression of the PKAcat subunit, however, markedly weak-

ened this interaction (Kd,EFF = 145,231 ± 3,084). By contrast, coexpression

of the PKAcat subunit in cells expressing fluorophore-tagged β2B and 4-SA

mutant Rad had no effect on FRET binding (Kd,EFF = 4,349 ± 138 versus

Kd,EFF = 4,346 ± 197 with and without, respectively, the PKA catalytic

domain; Fig. 4c and Extended Data Fig. 9c). These results suggest that

phosphorylation of Rad is required for dissociation of the Rad–β2B inter-

action. In a similar manner, PKA phosphorylation of Rad also reduced

FRET binding to both β 3 and β 4 (Extended Data Fig. 9a–c).

Unified mechanism for PKA regulation

In adrenal chromaffin cells and the sinus node cells of the heart, L-type

CaV1.3 channels are robustly stimulated by PKA^32 ,^33. We found that, in

HEK293T cells transfected with only CaV1.3 α1D plus β2B, superfusion of

forskolin over 1–3 minutes had no impact on the Ba2+ current (Fig. 4d–f).

By contrast, in cells expressing α1D, β2B and Rad, applying forskolin

increased Gmax by as much as 2.3-fold and by a mean of 1.9-fold, and

shifted the V 50 for activation (Fig. 4d–f). We also expressed the N-type

CaV2.2 α1B subunit, which is widely expressed in neurons, with β2B and

Rad in HEK293T cells. Forskolin increased Gmax through CaV2.2 when

coexpressed with Rad by a mean of 2.2-fold and shifted the V 50 for activa-

tion (Fig. 4g–i). For both CaV1.3 and CaV2.2, attenuating binding of Rad

to β subunits prevented the forskolin-induced modulation of CaV2.2 cur-

rent (Extended Data Fig. 8e, f ). We also expressed in HEK293T cells the

CaV2.2 α1B subunit with β2B and Rem, another member of the RGK GTPase

family. Forskolin increased Gmax through CaV2.2 when coexpressed with

Rem by 1.6-fold and shifted the V 50 for activation (Fig. 4g–i). Thus, PKA-

mediated modulation of CaV channels is not idiosyncratic, as currently

believed; rather, it is emerging to be a universal mechanism transferable

to all CaV channels that bind β subunits.

Discussion

The core α1C and β2B subunits, previously hypothesized to contain the

PKA target sites required for β-adrenergic agonist-induced stimulation

of CaV1.2, do not. Rather, successful reconstitution of regulation in a

heterologous expression system requires an additional component,

which we now identify as Rad. The cAMP–PKA-mediated regulation

of CaV1.2 requires both phosphorylation by PKA on the C terminus of

Rad and the interaction of Rad with the β subunit. Multiple-alignment

analysis of Rad from mice and other species shows that the four phos-

phorylation sites are conserved (Extended Data Fig. 10a). The required

interaction with the β subunit is consistent with our recent finding that

disrupting the α1C–β interaction prevents the regulation of CaV1.2 by

PKA in the heart^17.

Analysis of Rad and other members of the RGK GTPase family indi-

cates that their C-terminal phosphorylation sites are highly similar

(Extended Data Fig. 10b). Short stretches of basic and hydrophobic

amino acids are known to interact with the membrane^27 and phospho-

rylation of residues within these stretches alters their electrostatic

character, thereby reducing membrane affinity^34. We found that phos-

phorylation of two serine residues within the C-terminal polybasic

region of Rad releases Ca2+ channels from Rad-mediated inhibition,

probably by means of reducing the affinity of Rad with the membrane

and with the CaV β subunit (Fig. 4j, k). This mechanism of regulation is

modular and transferable, as CaV1.3 channels and neuronal CaV2.2 chan-

nels are also imparted with forskolin/PKA-mediated upregulation via

Rad and Rem. The activation of Ca2+ channels via release of inhibition

0

–5

–10

–15

–20

–25

4.0

3.0

2.0

1.0

0

βreceptor-adrenergic

Basal

+++++Rad

α 1

β 2

AC

0

–30

–40

–50

–60

a b

c

d e f

g

h i

j k

Forskolin:–+

–+ –+ –+

–+

Forskolin:

50 ms

Forskolin

Forskolin

Forskolin

50 ms

50 ms

50 ms

50 ms

5 pA pF

–1

(2 mM Ba

2+)

5 pA pF

–1

(2 mM Ba

2+)

1 pA pF

–1

(10 mM Ba

2+)

5 pA pF

–1

(10 mM Ba

2+)

5 pA pF

–1

(10 mM Ba

2+)

Rad:

`2B: WT WT Mut

WT Mut WT

5

4

3

2.5

2.0

1.5

1.0

0.5

0

2.5

2.0

1.5

Rad:

Rad Rad

**** ****

RemRem

No Rad:

No No

WT No WT

1.0

0.5

0

Ca [Ven–4SA Rad]free

V1.3 CaV1.3

CaV2.2 CaV1.3

CaV2.2

*

***

CaV2.2

_1D + `2B

_1D + `2B+ Rad

_1B + `2B+ Rad

_1B + `2B

_1B + `2B+ Rem

Ven–WT Rad

Ven–4SA Rad

0.4

0.3

0.2

0.1

0

0.5

0.5

0.4

0.3

0.2

0.1

0

0 1 × 105

1 × 105

Control

0

PKA

[Ven–Rad]free

CaV1.2

Control

PKA

Fold change

Gmax

(forskolin/no forskolin)

Fold change

Gmax

(forskolin/no forskolin)

Fold change

Gmax

(forskolin/no forskolin)

V^50

(mV)

++

β-agonist

Ca2+ Ca2+

Ca2+

PKA

G protein

ATPcAMP

++ Stimulated

Rad

+++

+++

Rad

α 1

β 2

V^50

(mV)

Cer–β2B

β2B

β2B

Cer–β2B

ED

ED

+++

Rad +

Cer

Cer

+++

Ven Rad +

Ven

FRET

pairs

FRET

pairs

****

******

AC

Fig. 4 | RGK GTPases confer adrenergic regulation to CaV1.2, CaV1.3 and
CaV2.2 channels via binding to β. a, Fold change in Gmax for different
combinations of wild-type and mutant Rad and β2B subunit. Data are
mean ± s.e.m.; P < 0.0001 by one-way ANOVA; *P < 0.001 by Dunnett’s test.
The data for WT Rad and WT β2B are as in Fig. 3g. n = 76, 20 and 15, left to right.
Specific P values can be found in the associated Source Data (see
Supplementary Information). b, c, At the left are shown schematics of the FRET
pairs, Cer–β2B with Ven–WT-Rad (b) or Ven–4SA-mutant Rad (c) (where Ven is
the venus tag and Cer is the cerulean tag). At the right, FRET efficiency (ED) is
plotted against the free concentration of Ven–WT (b) or Ven–4SA-mutant Rad
(c). The solid line fits a 1/1 binding isotherm. d, Ba2+ current of CaV1.3 channels,
without or with expression of Rad, elicited by voltage ramp every 10 s. Black
traces indicate before and blue traces after forskolin treatment. No Rad, Rad:
seven cells each. e, Fold change in Gmax. Data are mean ± s.e.m.; ***P < 0.0001 by
unpaired two-tailed t-test; n = 7 in both cases. f, Boltzmann function parameter
V 50. Data are mean ± s.e.m.; P < 0.05; P < 0.01 by paired two-tailed t-test; n = 7
in all cases. g, Ba2+ current of CaV2.2 channels, without or with expression of Rad
or Rem, elicited by voltage ramp every 10 s. Black traces were obtained before,
and blue traces after, treatment with forskolin. Representative of 11, 11 and 15
cells, from top to bottom. h, Fold change in Gmax. Data are mean ± s.e.m.
P < 0.001 by one-way ANOVA; P < 0.001, P < 0.05 by Dunnett’s test. n = 11, 11
and 15, from left to right. i, Boltzmann function parameter V 50. Data are
mean ± s.e.m.; **P < 0.001 by paired two-tailed t-test; n = 11, 11 and 15, from left
to right. j, k, Proposed model of β-adrenergic regulation of Ca2+ channels.
j, k, Basal state (j), and after β-agonist-induced activation of adenylyl cyclase
(AC) (k) leads to activation of PK A and hence to PK A-mediated phosphorylation
of Rad, causing dissociation of Rad from the CaV1.2 complex and therefore
increased Ca2+ inf lux.