Identification of apocalmodulin and Ca2+-calmodulin regulatory domain in skeletal muscle Ca2+ release channel, ryanodine receptor.
Fusion proteins and full-length mutants were generated to identify the Ca(2+)-free (apoCaM) and Ca(2+)-bound (CaCaM) calmodulin binding sites of the skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1). [(35)S]Calmodulin (CaM) overlays of fusion proteins revealed one potential Ca(2+)-dependent (aa 3553-3662) and one Ca(2+)-independent (aa 4302-4430) CaM binding domain. W3620A or L3624D substitutions almost abolished completely, whereas V3619A or L3624A substitutions reduced [(35)S]CaM binding to fusion protein (aa 3553-3662). Three full-length RyR1 single-site mutants (V3619A,W3620A,L3624D) and one deletion mutant (Delta4274-4535) were generated and expressed in human embryonic kidney 293 cells. L3624D exhibited greatly reduced [(35)S]CaM binding affinity as indicated by a lack of noticeable binding of apoCaM and CaCaM (nanomolar) and the requirement of CaCaM (micromolar) for the inhibition of RyR1 activity. W3620A bound CaM (nanomolar) only in the absence of Ca(2+) and did not show inhibition of RyR1 activity by 3 microm CaCaM. V3619A and the deletion mutant bound apoCaM and CaCaM at levels compared with wild type. V3619A activity was inhibited by CaM with IC(50) approximately 200 nm, as compared with IC(50) approximately 50 nm for wild type and the deletion mutant. [(35)S]CaM binding experiments with sarcoplasmic reticulum vesicles suggested 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 binding domain that is shared by apoCaM and CaCaM.
Yamaguchi
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N
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Xin
C
C
Meissner
G
G
D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D002147:Calmodulin; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D013462:Sulfur Radioisotopes; D002118:Calcium