N. Yamaguchi et al., Identification of apocalmodulin and Ca2+-calmodulin regulatory domain in skeletal muscle Ca2+ release channel, ryanodine receptor, J BIOL CHEM, 276(25), 2001, pp. 22579-22585
Fusion proteins and full-length mutants were generated to identify the Ca2(
+)-free (apoCaM) and Ca2(+)bound (CaCaM) calmodulin binding sites of the sk
eletal muscle Ca2+ release channel/ryanodine receptor (RyR1). [S-35]Calmodu
lin (CaM) overlays of fusion proteins revealed one potential Ca2+-dependent
(aa 3553-3662) and one Ca2+-independent (aa 4302-4430) CaM binding domain.
W3620A or L3624D substitutions almost abolished completely, whereas V3619A
or L3624A substitutions reduced [S-35]CaM binding to fusion protein (aa 35
53-3662). Three full-length RyR1 single-site mutants (V3619A, W3620A,L3624D
) and one deletion mutant (Delta 4274-4535) were generated and expressed in
human embryonic kidney 293 cells. L3624D exhibited greatly reduced [35S]Ca
M binding affinity as indicated by a lack of noticeabIe binding of apoCaM a
nd CaCaM (nanomolar) and the requirement of CaCaM (micromolar) for the inhi
bition of RyR1 activity. W3620A bound CaM (nanomolar) only in the absence o
f Ca2+ and did not show inhibition of RyR1 activity by 3 muM CaCaM. V3619A
and the deletion mutant bound apoCaM and CaCaM at levels compared with wild
type. V3619A activity was inhibited by CaM with IC50 similar to 200 nM, as
compared with IC50 similar to 50 nM for wild type and the deletion mutant.
[S-35]CaM binding experiments with sarcoplasmic reticulum vesicles suggest
ed that apoCaM and CaCaM bind to the same region of the native RyR1 channel
complex. These results indicate that the intact RyR1 has a single CaM bind
ing domain that is shared by apoCaM and CaCaM.