DIFFERENTIAL ACTIVATING AND DEACTIVATING EFFECTS OF NATURAL RYANODINECONGENERS ON THE CALCIUM-RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM - EVIDENCE FOR SEPARATION OF EFFECTS AT FUNCTIONALLY DISTINCT SITES
Ra. Humerickhouse et al., DIFFERENTIAL ACTIVATING AND DEACTIVATING EFFECTS OF NATURAL RYANODINECONGENERS ON THE CALCIUM-RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM - EVIDENCE FOR SEPARATION OF EFFECTS AT FUNCTIONALLY DISTINCT SITES, Molecular pharmacology, 44(2), 1993, pp. 412-421
Two novel natural ryanoids from extracts of the wood of Ryania specios
a Vahl were evaluated with sarcoplasmic reticulum (SR) vesicles for th
eir binding affinities and their activating and deactivating effects o
n Ca2+ release channels. The new ryanoids, which are more polar than t
he known Ryania constituents ryanodine and didehydro-(9,21)-ryanodine,
were purified using silica gel column chromatography and reverse phas
e high performance liquid chromatography. The new ryanoids were design
ated ester E and ester F, in keeping with nomenclature previously used
in the literature. These compounds were identified by NMR spectroscop
y and mass spectroscopy as C9ax-hydroxyryanodine and C8ax-hydroxy-C-10
-epi-dehydroryanodine, respectively. Binding of esters E and F to the
high affinity (nanomolar K(d)) site on SR Ca2+ release channels was de
termined from relative binding affinity assays using 6.7 nm [H-3]ryano
dine. Apparent K(d) values of ryanodine, ester E, and ester F for bind
ing to this domain on the skeletal muscle ryanodine receptor/SR Ca2+ r
elease channel were 4.4 +/- 0.8, 65 +/- 10, and 257 +/- 53 nM, respect
ively (mean +/- standard deviation, four or more experiments). Apparen
t K(d) values for cardiac muscle receptors were 0.51 +/- 0.01, 12 +/-
0.4, and 57 nm, respectively. As a functional indication of the effect
s of the ryanoids, channel-opening (activator) and channel-closing (de
activator) actions were assessed from the ability of the ryanoids to a
lter the rate of Ca2+ efflux from passively loaded skeletal muscle jun
ctional sarcoplasmic reticular vesicles (JSRV). Activator actions amon
g the ryanoids were similar, in that they exhibited apparently paralle
l concentration-effect curves, having a slope of 40% Ca2+ loss/decade
increment in ryanoid concentration. Half-maximal values for activation
(EC50 values) were 2.5, 63, and 43 muM for ryanodine, ester E, and es
ter F, respectively. Maximal channel opening by ester E was significan
tly less than that produced by the other ryanoids. The deactivator act
ions of the compounds on skeletal JSRV were dissimilar, in that their
concentration-effect curves appeared not to be parallel. The quotient
of the EC50 for deactivation and that for activation was taken as the
concentration-coupling ratio (CCR). The CCR for ryanodine was 114 and
that for ester F was 72, but the CCR for ester E was only 21. ATP-depe
ndent Ca2+ accumulation by cardiac JSRV provided a second means to eva
luate deactivator actions of the ryanoids. Results from cardiac JSRV a
ssays were in general similar to those from skeletal JSRV assays. CCRs
among the natural ryanodine analogs were obviously disparate, providi
ng strong evidence for separation of ryanoid effectiveness at function
ally distinct domains on (or states of) the ryanodine receptor/SR Ca2 release channel. In support of this conclusion, results of studies wi
th the parent molecule ryanodine also revealed a variant CCR between h
alf-maximally deactivating and half-maximally activating concentration
s. In the presence of the allosteric channel activator adenosine-5'-be
ta,gamma-methylenetriphosphate, ryanodine exhibited an activation curv
e that lay >3 orders of magnitude to the left of its deactivation curv
e. The CCR of ryanodine in the absence of this adenine nucleotide was
only 2% of that in its presence. The observation that activation is no
t invariantly concentration-coupled to deactivation suggests that the
molecular features responsible for the activator actions of ryanoids o
n Ca2+ release channels are different from those inducing deactivation
of the channels. The present results constitute the first evidence th
at the activator effects of ryanoids are functionally separable from t
heir deactivator effects.