Molecular and Cellular Biochemistry 114: 119-123, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca 2+ release channel Gerhard Meissner and Anita E1-Hashem Departments of Biochemistry and Biophysics, and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA

Abstract Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca 2+ release channel (ryanodine receptor) in excitable tissues. Using [3H]ryanodine, a 30 S protein complex comprised of four polypeptides of Mr 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [3H]ryanodine binding and Ca 2+ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel. (Mol Cell Biochem 114: 119-123, 1992)

Key words: ryanodine receptor, Ca 2+ release channel, sarcoplasmic reticum, excitation-contraction coupling

Introduction Ryanodine is a neutral plant alkaloid which, depending on muscle type and activity, causes contracture or a decline in contractile activity [1]. [3H]Ryanodine has had a major impact on investigation of the mechanism of excitation-contraction coupling having been utilized as a probe in the isolation and subsequent cloning of a 30 S sarcoplasmic reticulum (SR) protein complex comprised of four 565 kDa polypeptides (the ryanodine receptor) [2, 3]. The isolated receptor contains an intrinsic Ca 2+ channel activity which is regulated by various effector molecules including Ca 2+, Mg 2+ and ATP. Morphological studies have shown that the SR ryanodine receptor/Ca z+ release channel is identical with large protein structures ('feet') which, in skeletal muscle, are located at specialized junctions where the trans-

verse tubular and SR membranes become closely apposed to form a narrow 12 nm gap. In this article, we summarize the results of our research focused on understanding the interaction of ryanodine with the SR Ca 2+ release channel. Our studies suggest that SR Ca 2+ release channel activity and [3H]ryanodine binding are dependent on ionic strength and affected in a similar manner by divalent cations.

Results and discussion The functional consequences of the interaction of ryanodine with the SR Ca 2+ release channel have been most convincingly demonstrated in single channel recordings, using the planar lipid bilayer technique [4, 5].

Address for offprints: G. Meissner, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA

120

~,~,..~1~., +30 pM Ryonodine

c -

.~ 3.0

E

O--

U')

+2 mMRyanodin~,

I..LI ._l

Cm CO ILl .... ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...............................

>

J

I'ODO ms

CO

0 m

Effect of ryanodine on a single reconstituted, purified rabbit skeletal muscle Ca 2÷ release channel. Shown are single-channel recordings of K + current of a purified 30 S channel complex incorporated into a planar lipid bilayer in symmetric 250 mM KC1 buffer with 50/xM free Ca 2+. Unitary conductance = 700pS. Holding potential = 20 mV. Upper trace shows the appearance of a subconducting state with open probability (Po) - 1 following several rain after cis addition of 30 I~M ryanodine. An additional, infrequently observed substate also can be observed. Lower trace illustrates the sudden transition from the subconductance state to a fully closed state within one min after cis addition of 2 mM ryanodine. Bars on left represent the open (o) and closed (c) channel (taken with permission from ref. 5). F i g . 1.

Figure 1 shows a single K + conducting skeletal muscle C a 2+ release channel which was recorded following the addition of the purified, Chaps-solubilized channel complex to the cis side of the bilayer (defined as the side of the bilayer to which the channel is added; cis side corresponds to the SR cytoplasmic side). Micromolar concentrations of ryanodine induced the formation of a channel state characterized by long open intervals and a reduced conductance (upper trace of Fig. 1). Subsequent addition of millimolar concentrations of ryanodine completely closed the channel. Both the subconducting and closed channel states became insensitive to regulation by endogenous effector molecules such as C a 2+, Mg 2+ or ATP. Thus, ryanodine may 'lock' the SR Ca 2+ release channel into an open or closed state. Ryanodine binds slowly to the SR Ca :+ release channel, whereas the lifetime of planar lipid bilayers is limited (generally less than 1 h). It is therefore often more practical to study the pharmacological effects of ryanodine using isolated 'heavy' (junctionally derived) SR vesicle fractions [2]. In Fig. 2, 45CaZ+loaded SR vesicles were incubated for 45 min with varying concentrations of ryanodine. The 45Ca2+ release behavior of the vesicles was then studied in media which either inhibited or activated the ryanodine-unmodified SR Ca 2+ release channel. In the absence of ryanodine, vesicles retained t h e i r 45Ca2+ stores where transferred to a medium that

1.0

z ÷ N

o o

~o

o

0 .[.-~j

I

I

I

I

I

I

0

0.001

0.01

0.1

I

I0

I00

RYANODINE

Fig. 2. Dependence of

(~.IM)

effiux on ryanodine concentration. 'Heavy' skeletal muscleSR Ca2+release vesicles were incubated for 45 min at 37°C with 0.1 mM free 45Ca2+and the indicated concentrations of ryanodine. Amounts of 45Ca2+retained by the vesicles in Ca2+ release channel inhibiting (10mM Mg2+, 10txM ruthenium red (RR, 0)) and activating(5/xM free Ca2+ (O)) media were determined by filtration (taken with permissionfrom ref. 9). 45Ca2+

contained the two channel inhibitors M g 2+ and ruthenium red (RR). In contrast, vesicles released a major portion of their 45Ca2+ stores when the SR Ca 2+ release channel was activated by transferring the vesicles to a 5/,M Ca :+ release medium. Incubation with nanomolar concentrations of ryanodine rendered the channel insensitive to inhibition by Mg z+ and ruthenium red, which indicated that ryanodine had locked the channel into an open configuration (Fig. 2, upper trace). At concentrations above 10/,M, nearly maximal amounts o f Ca 2+ were retained by the vesicles in both the channel inhibiting and activating medium, which suggested that ryanodine had locked the channel into a fully closed state (Fig. 2, lower trace). [3H]Ryanodine binding studies have indicated that ryanodine exerts its dual action by binding to high- and low-affinity channel sites (ref. 2, Fig. 3). Comparison of the number of high affinity sites obtained by Scatchard plot analysis (inset of Fig. 3) and total number of binding sites suggests that for each high affinity site there are two to three low affinity sites. The extent of occupancy of the low affinity ryanodine sites to effect channel closure is not known, however. High-affinity ryanodine binding has been shown to be sensitive to Ca 2+, Mg z+ and adenine nucleotides, and enhanced by increasing the ionic strength of the assay media [2]. Table I summarizes the results of experi-

121 ments in which 45Ca2+ effiux rates and amounts of [3H]ryanodine bound to SR vesicles were determined at three different free Ca 2+ concentrations using two media of differing ionic strength (0.1 or 1.0 M KC1). As previously observed [6], 45Ca2+ release from SR vesicles was faster in the 0.1M KC1 medium at 2.5 10-5M free Ca 2+ than at < 10 -8 or 10 -3 M free Ca 2+. [3H]ryanodine binding displayed an essentially identical C a 2+ dependence. Binding was highest at the micromolar Ca 2+ concentrations, and decreased as the free Ca 2+ concentration was lowered to nanomolar or increased to millimolar concentrations. 45Ca2+ efflux rates and [3H]ryanodine binding increased at all three Ca 2+ concentrations as the KC1 concentration was raised from 0.1 to 1.0M. The most dramatic effects were observed at 10 -3 M free Ca z+ where a tenfold increase in ionic strength resulted in a greater than 25-fold increase in ryanodine binding and Ca 2+ release channel activity. Data of Table 1 support the idea that ryanodine binding is enhanced under conditions than favor 'opening' of the SR Ca 2+ release channel [2]. Dependence of SR Ca 2+ release channel activity and [3H]ryanodine binding activities o n C a 2+ concentration suggests that the channel possesses high-affinity activating and low-affinity inhibitory Ca 2+ binding sites [2]. The divalent cation specificity of these sites was tested by determining the amounts of ryanodine bound to SR vesicles in media that contained 0.1 M KC1 and varying concentrations of either Ca z+, Sr 2+, Mg 2+ or Ba 2+ (Fig. 4). Increase in free Ca 2+ from 10/xM to 60/xM or addition of 75/xM Sr 2+ to the 10/xM C a 2+ medium resulted in increased ryanodine binding. Both Mg 2+ and Ba 2+ inhibited ryanodine binding. At concentrations above 1 mM, all four divalent cations had a strongly inhibitory effect. These results suggest that the high affinity Ca 2+

"-6 6O

2.o~,

6

g

n I

I\

4o Z

BOUND

o z< ~2o g2

20 o

to

z o

o

I° 10-9

I i 0 -m

I

i0 -r

I i 0 -~

I

i0 -5

FREE[3HIRYANODINE(M) Fig. 3. High and low affinity ryanodine binding to 'heavy' skeletal muscle SR vesicles. Seatchard analysis (inset) reveals a curvilinear slope indicating the presence of both high affinity (KD = 7nM) and low-affinity sites. Specific [3H]ryanodine binding to SR membranes was determined as described [5].

binding site has an apparently narrow divalent cation specificity, Sr 2+ being the only other divalent cation investigated capable of activating the channel. By comparison, a broad divalent cation specificity was observed for the low-affinity inhibitory Ca 2+ binding site(s). Single channel measurements have shown that, in addition to Ca 2+, the SR Ca 2+ release channel conducts Ba 2+ and Mg 2+ and remains active with 50 mM C a 2+, Mg 2+ or Ba 2+ in the trans chamber [7, 8]. Since it was conceivable that a lumenal regulatory, divalent-cation binding protein was lost in the bilayer experiments, we also determined sidedness of channel inhibition by divalent cations with the use of intact SR vesicles. Fig. 5 illustrates the time courses of 45Ca2+ release from SR vesicles which were passively loaded with 1 mM 45Ca2+ in media containing and lacking 5raM Ba2+~ In the

Table 1. The effect of Ca z+ and ionic strength on SR Ca 2+ release channel activity and [3H]ryanodine binding [KCI] (M)

[Ca 2+] (M)

45Ca2+ release T1/2 (s)

[3H]Ryanodine binding (pmol/mg)

0.1

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel.

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca2+ release channel (ryanodine receptor) in excitable tissues. ...
336KB Sizes 0 Downloads 0 Views