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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
47
25-POS
Board 25
Effect of Camkii-Mediated Phosphorylation in the Β
1a
Subunit of the Voltage-Gated Ca
2+
Channel.
Dora Bodnar
1
, Claudio F. Perez
1
,
Jose M. Eltit
2
.
2
Virginia Commonwealth University, School of Medicine, Richmond, VA, USA.
1
Brigham and
Women's Hospital, Harvard Medical School, Boston, MA, USA,
The β
1a
subunit is part of the DHPR-RyR1 complex in skeletal muscle. This macro-protein
complex transduces electrical signals in the plasma membrane into cytosolic Ca
2+
transients,
triggering muscle contraction. We showed that mutations in
489
QVQVLTSLRRNLSFW
503
C-
term tail domain of β
1a
hinder the conformational communication between the DHPR and the
RyR1. The residue Ser
501
is predicted to be a phosphorylation target for various protein kinases
(motif NLSFW). In this study, we evaluated the specificity of protein kinases to phosphorylate
this site
in vitro
and we further evaluate its effects on calcium handling in cultured myotubes.
The C-term tail of β
1a
subunit, encompassing residues V
485
through M
524
, was expressed as a
GST-fusion protein and subjected to
in vitro
phosphorylation by PKC, MAPK and CaMKII.
Western blot analysis with an anti-phospho antibody confirmed specific phosphorylation of the
C-term domain by CaMKII but not PKC or MAPK. Alanine substitution of residue S
501
(S501A
mutation) further prevented CaMKII phosphorylation confirming S
501
as the phosphorylation
site. The effect of phosphorylation on Ca
2+
handling was evaluated in β
1
-null myotubes stably
transfected with β
1a
subunit carrying mutation S501A. Patch-clamp analysis of wt and S501A
myotubes showed small to no difference in the L-type Ca
2+
current between both genotypes.
However, Ca
2+
imaging studies revealed a significant increase in the amplitude of
depolarization-induced Ca
2+
transient (K
+
stimulation) in myotubes expressing the S501A
mutation. In addition, mutant cells displayed significantly higher levels of SR Ca
2+
content and
faster rate of decay of the depolarization-induced Ca
2+
transient response. Our findings suggest
that residue S
501
of β
1a
subunit is a substrate for CaMKII phosphorylation and that its state of
phosphorylation modulates Ca
2+
release in skeletal muscle cell, presumably altering the DHPR to
RyR1 signaling.
Supported by grant NIH-1R01AR06773