-Adrenergic Stimulation of L-type Ca
2
Channels in
Cardiac Myocytes Requires the Distal Carboxyl
Terminus of
1C
but Not Serine 1928
Anand N. Ganesan, Christoph Maack, David C. Johns, Agnieszka Sidor, Brian O’Rourke
Abstract—-Adrenoceptor stimulation robustly increases cardiac L-type Ca
2+
current (I
CaL
); yet the molecular mechanism
of this effect is still not well understood. Previous reports have shown in vitro phosphorylation of a consensus protein
kinase A site at serine 1928 on the carboxyl terminus of the
1C
subunit; however, the functional role of this site has
not been investigated in cardiac myocytes. Here, we examine the effects of truncating the distal carboxyl terminus of
the
1C
subunit at amino acid residue 1905 or mutating the putative protein kinase A site at serine 1928 to alanine in
adult guinea pig myocytes, using novel dihydropyridine-insensitive
1C
adenoviruses, coexpressed with
2
subunits.
Expression of
1C
truncated at 1905 dramatically attenuated the increase of peak I
CaL
induced by isoproterenol. However,
the point mutation S1928A did not significantly attenuate the -adrenergic response. The findings indicate that the distal
carboxyl-terminus of
1C
plays an important role in -adrenergic upregulation of cardiac L-type Ca
2+
channels, but that
phosphorylation of serine 1928 is not required for this effect. (Circ Res. 2006;98:e11-e18.)
Key Words: protein kinase A
adenovirus
ion channel
calcium current
cAMP
V
oltage-gated L-type (Ca
v
1.2) Ca
2+
channels play a central
role in controlling cardiac function, providing the trigger
for intracellular Ca
2+
release during excitation– contraction cou-
pling, contributing to the plateau phase of the cardiac action
potential, and modulating pacemaker activity in the sinoatrial
node.
1–3
The L-type Ca
2+
channel is a multiprotein complex,
comprised of the pore-forming
1
subunit and the auxiliary
2
and
2
subunits.
4
In the heart, the molecular identity of the
subunit remains a subject of ongoing investigation.
5,6
A number of receptor-mediated signal transduction pathways
regulate Ca
2+
influx via Ca
v
1.2 channels, the most prominent of
which is the -adrenergic/cAMP signaling pathway.
-Adrenergic stimulation is the dominant mechanism of positive
chronotropy, inotropy, and lusitropy in the heart.
7
Activation of
-adrenergic receptors strongly enhances cardiac L-type Ca
2+
current (I
CaL
) via cAMP generation and the activation of protein
kinase A (PKA), presumably by phosphorylating specific sites
on the channel protein.
8,9
Nevertheless, the structural determi-
nants of PKA action on the Ca
2+
channel in native adult
cardiomyocytes are unknown.
Previous biochemical studies have implicated the carboxyl
terminus as a target of PKA-mediated phosphorylation: for
example, a truncated form of the channel was reportedly not
phosphorylated by PKA in vitro.
10
The consensus phosphor-
ylation site at serine 1928 was later identified as the main
target of PKA.
11,12
On the other hand, phosphorylation sites
have also been mapped to the subunit of the channel and
localized to serines 478 and 479 of
2a
.
13,14
Mixed results have been obtained when attempts have been
made to confirm the functional role of these sites in the
regulation of Ca
2+
channel activity, in part, because of the
difficulty of reconstituting PKA-mediated channel activation
in heterologous expression systems. Whereas some studies
have observed PKA-mediated activation of the channel in
heterologous systems,
10,14
others have observed no such
stimulation.
15,16
Other groups have used PKA inhibitors to
argue that Ca
v
1.2 channels are phosphorylated at baseline
when expressed in cultured cell lines.
17–19
The failure to reproduce the strong upregulation of the L-type
Ca
2+
current in nonnative cells has been attributed to the lack of
A-kinase anchoring proteins (AKAPs) in heterologous systems.
By coexpressing AKAP 79, Gao et al
20
were able to reconstitute
forskolin-mediated upregulation of Ca
2+
channels expressed in
HEK 293 cells, and the authors concluded that S1928 was
necessary to produce PKA-mediated activation of the L-type
current. In contrast, a subsequent study by the same group
reported PKA-mediated activation of Ca
2+
current through
L-type channels with a mutant
1C
truncated at 1905 and
Original received November 8, 2005; revision received December 15, 2005; accepted December 20, 2005.
From the Institute of Molecular Cardiobiology (A.N.G., C.M., A.S., B.O’R.), Department of Medicine; and the Department of Neurosurgery (D.C.J.), The
Johns Hopkins University, Baltimore, Md.
Under a licensing agreement between Excigen Inc and the Johns Hopkins University, D.C.J. is entitled to a share of royalty and milestone payments
received by the University on sales of products described in this article. The terms of this agreement are being managed by the Johns Hopkins University
in accordance with its conflict-of-interest policies.
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Brian O’Rourke, Johns Hopkins University, 1059 Ross Building, 720 Rutland Ave, Baltimore, MD 21205. E-mail bor@jhmi.edu
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000202692.23001.e2
e11
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