2
66
Jacobson et al.
the ubiquitous nature of these types of residues in proteins. Acknowledgments
However, the narrow range of pK values of the most potent
a
We are grateful to Ronit Hirsch for preparing the herpes simplex
-CmC analogs suggests the involvement of a general base such virus amplicons used for transduction of RyR cDNAs into 1B5 myo-
as a lysine or histidine. This hypothesis is supported by recent tubes.
4
work in our laboratory indicating that a mutation of a dipeptide
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4020
4021
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Lys
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Baur CP, Bellon L, Felleiter P, Fiege M, Fricker R, Glahn K, Heffron JJ, Herrmann-
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zation of the 1-hydroxyl of 4-CmC, although this hypothesis
requires further testing.
Putative hydrophobic binding sites that may interact with
chemical groups at the 3,4-positions of 4-CmC are more
readily identified. Within the 173 amino acid region of RyR1
required for 4-CmC activation (Fessenden et al., 2003), the
most hydrophobic region lies between amino acids 4025 to
Beeler TJ and Gable KS (1993) Activation of Ca2 release from sarcoplasmic retic-
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ϩ
2
ϩ
segment contains the putative “Ca
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4032
Glu
hand motifs implicated in Ca regulation of RyR1 activity
Xiong et al., 1998, 2006; Fessenden et al., 2004) (Fig. 9).
(Chen et al., 1998) and also is in proximity to EF
2ϩ
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(
Thus, this hydrophobic segment is an attractive region to
consider as a portion of the 4-CmC binding site, especially
2ϩ
because 4-CmC activates RyR1 via modulation of the Ca
sensitivity of the channel (Herrmann-Frank et al., 1996b).
An alternate possibility is that the 4-CmC binding site on
RyR1 lies near the lipid/protein interface and that the hydro-
phobic portions of the 4-CmC molecule anchor the compound
to the sarcoplasmic reticulum membrane. This concept is
supported by the fact that long-chain alkyl phenols are po-
tent activators of RyR1 and increasing the length of the alkyl
chain increases the potency of the compound (Beeler and
Gable, 1993). The hydrophobic segment between amino acids
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ϩ
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myogenic cell line lacking ryanodine receptor protein for homologous expression
studies: reconstitution of Ry1R protein and function. J Cell Biol 140:843–851.
4
025 to 4041 remains a good candidate for a 4-CmC binding
site, because it was originally predicted as transmembrane
helix (M2) of RyR1 (Zorzato et al., 1990). In this hypothetical
case, the hydrophilic segments required to interact with the
hydroxyl group on 4-CmC would be expected to be in prox-
imity to this helix. However, it should be noted that recent
experiments suggest that this segment does not constitute a
transmembrane helix, but instead is cytosolic (Du et al.,
2ϩ
Palade P (1987) Drug-induced Ca release from isolated sarcoplasmic reticulum.
III. Block of Ca2 -induced Ca
ϩ
2ϩ
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8
4:2655–2663.
2ϩ
Pessah IN, Stambuk RA, and Casida JE (1987) Ca -activated ryanodine binding:
mechanisms of sensitivity and intensity modulation by Mg2 , caffeine and adenine
nucleotides. Mol Pharmacol 31:232–238.
ϩ
Pessah IN, Waterhouse AL, and Casida JE (1985) The calcium-ryanodine receptor
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gene delivery system for skeletal muscle myoblasts and myotubes. Am J Physiol
2
002). If this is the case, this region could still be involved in
binding a lipid solvated 4-CmC molecule if it is in proximity
to the surface of the lipid bilayer.
278:C619–C626.
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2ϩ
fication of a two EF-hand Ca binding domain in lobster skeletal muscle ryano-
dine receptor/Ca2 release channel. Biochemistry 37:4804–4814.
ϩ
2ϩ
Conclusions
Xiong L, Zhang JZ, He R, and Hamilton SL (2006) A Ca -binding domain in RyR1
that interacts with the calmodulin binding site and modulates channel activity.
Biophys J 90:173–182.
Our work suggests that the structural features of phenolic
compounds required to activate RyR1 include an ionizable pro-
Zorzato F, Fujii J, Otsu K, Phillips M, Green NM, Lai FA, Meissner G, and MacLen-
nan DH (1990) Molecular cloning of cDNA encoding human and rabbit forms of the
2ϩ
ton (pK ϭ 8–10.5) at the 1-position and hydrophobic groups at
Ca release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticu-
lum. J Biol Chem 265:2244–2256.
a
the 3,4- and/or 5-positions of the benzene ring. In addition,
phenols with highly hydrophobic groups are more potent acti-
vators of RyR1 compared with 4-CmC (the most potent com-
pound we tested is compound 5b (with an EC50 ϭ 31 M).
Finally, the results with compound 6b are encouraging in that
this compound offers a chemical “handle” whereby a suitable
reporter group can potentially be linked to identify the region(s)
within the protein where the 4-CmC analogs bind.
Zorzato F, Scutari E, Tegazzin V, Clementi E, and Treves S (1993) Chlorocresol: an
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ϩ
1
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2ϩ
Zucchi R and Ronca-Testoni S (1997) The sarcoplasmic reticulum Ca
channel/
ryanodine receptor: modulation by endogenous effectors, drugs and disease states.
Pharmacol Rev 49:1–51.
Address correspondence to: Dr. James D. Fessenden, Boston Biomedical
Research Institute, 64 Grove St., Watertown, MA 02472. E-mail: fessenden@
bbri.org