Charged surface area of Maurocalcine determines its interaction with the skeletal muscle calcium release channel

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In this work we evaluated how the replacement of different amino acids in the sequence of the wt MCa affects its ability to modify the gating of the skeletal type RyR. We show that mutations of MCa within the cluster of negatively charged residues surrounding ²⁴Arg decrease the appearance of long-lasting subconductance states on isolated channels reconstituted into planar lipid bilayers and long-lasting embers in saponin-treated adult striated muscle fibers. The extent of this effect depended on the direction of the current in lipid bilayer experiments indicating a voltage dependent binding of the toxin to the channel.

Effect of mutations in MCa on the lengths of LLSS and ECRE

In previous studies we have demonstrated that MCa increases the frequency of ECRE together with a decrease in the amplitude of the individual events in saponin-treated adult mammalian striated muscle fibers (Szappanos et al., 2005). In addition, it induces long-lasting embers with durations usually exceeding 200 ms, occasionally lasting longer than 1.5 s. On purified RyR1 reconstituted into planar lipid bilayers, the toxin induced both an increase in the open probability of the channel and the appearance of long-lasting open events characterized by a reduced conductance (Chen et al. 2003; Estève et al. 2003). As a consequence of these effects, MCa caused a dramatic increase in the [³H]ryanodine binding to RyR1 as well as in Ca²⁺ release from SR vesicles. All these effects were completely abolished by the point mutation of the Arg residue in position 24 of MCa (Estève et al. 2003; Szappanos et al., 2005) supporting the hypothesis that the basic amino acid-rich region is important for the functional effect of MCa on RyR1.

Here we show that the effects of MCa described for LLSS and for ECRE depend on the position of mutation of the toxin and correlate with the distance of the mutation from the charged surface of MCa. Indeed, the effects of [Ala⁸]MCa and [Ala¹⁹]MCa mutants on ECRE were almost identical to the wt toxin, in the presence of [Ala²²]MCa the length of embers decreased, and finally the [Ala²⁴]MCa analogue did not show any effect on ECRE. These results are in direct agreement with the observations originating from lipid bilayer experiments with these analogues illustrating that the capability for inducing the long-lasting sub-conductance state of RyR1 depends on the place of mutation.

Changing amino acids at different positions – by substituting a charged amino acid with a neutral one – thus reveals that the dramatic effect of the mutations within the cluster of basic amino acids cannot be solely attributed to the change of the net electrical charge of the peptide since mutations distant to the cluster but producing the same net electrical charge had relatively minor effects. Since structural changes within MCa – due to its triple disulphide bonds – are unlikely to accompany the mutations, this cluster must represent the interacting site with the RyR.

The properties of the MCa binding site on RyR

Recent evidence has positioned the MCa binding site to residues 3351–3507 on RyR1 (Altafaj et al. 2005). The results presented here suggest that the toxin binding site is distant from the high affinity calcium binding (regulatory) site because the effects of the toxins were calcium independent. In addition, it seems also to be distinct from the ryanodine binding site (see below). Finally, it should be noted that the calcium release channel was still ohmic after binding the toxin or its mutants indicating an allosteric modification of the RyR conformation by the toxins.

The fact that the LLSS length was found to be independent of the concentration of MCa – or of the concentration of its analogues – suggests that one MCa binds to a RyR monomer. On the other hand, since the inter LLSS open probability is higher (by approximately 10 fold) than under control conditions indicates that the binding of 1, 2, or 3 MCa to the tetrameric channel preconditions its gating.

Given the above constrains, and assuming that MCa and its mutants bind to RyR1 via a common structural motif, then corresponding mutations should evoke parallel changes in the affinity if the continuity of the basic surface is essential, probably because the electric field-induced orientation of the toxin plays an important role in its binding to the channel. Since the continuity of the charged surface cluster seems equally – if not more – important than the charge itself, we conclude that the orientation of the peptide is the most important factor in determining the binding of the toxin. This suggests that the MCa binding site is near to the opening of the channel pore – where the electric gradient is extremely large – and thus the electric momentum dominates the orientation of the MCa molecule. In the above framework, due to the necessity of a suitable orientation of the charged surface, the interaction of MCa with the RyR should be influenced by the polarity of the membrane potential. In line with the above reasoning we found that the dissociation constants of the peptides were polarity dependent.

We have recently suggested that in intact cells MCa binds preferably to the open conformation of the RyR (Pouvreau et al. 2006). It should be noted that this conclusion was derived from comparing the prolonged calcium release after repolarisation to that of maximal calcium release during the depolarizing pulse. This prolonged calcium release should correspond to the LLSS state seen in lipid bilayer experiments. This suggests that the binding site for the toxin which is responsible for the induction of LLSS is not, or is less accessible when the channel is closed. Clearly, a binding site within or in close association to the channel pore would fulfill this criteria. If the binding site is indeed found within the pore the movement of positively charge ions through the channel (the calcium release process) should hinder the binding of the positively charged toxin. A similar phenomenon is expected to take place in bilayer experiments if the holding potential is set to drive the charge carrying ions in the physiological direction. This is indeed what was observed strengthening the idea that the binding site is somehow associated with the pore itself.

Combined effect ryanodine and MCa

Normally low concentrations of ryanodine induce a characteristic “half conductance” state of RyR from which the channel only occasionally closes, and only for a short time (for a few milliseconds), and never switches to the full open state unless the ryanodine is removed from the bathing solution (for review see Meissner and el-Hashem, 1992). In the case of the simultaneous presence of ryanodine and MCa the “half conductance state” characteristic to the alkaloid dominates channel behavior. Nevertheless, long closing periods – due to the presence of MCa – become apparent, during which the channel enters a “half of the half” conductance state clearly indicating that the binding of the toxin and the alkaloid are independent of one another. The free gating of the channel between these two states ensures that not only the binding but also the state into which MCa transforms the channel is independent of that induced by ryanodine.

The binding site for MCa and its effect on ECRE

MCa and its structural analogue Imperatoxin A have been shown to induce extremely long calcium release events in amphibian skeletal muscle fibers (Gonzalez et al. 2000b; Stifmann et al. 2000; Szappanos et al. 2005). These events are most likely the direct consequence of the calcium release channel entering into the LLSS following the binding of the toxin molecule. Several pieces of evidence support this conclusion. First, the length of these long events correlates well with the length of LLSS. Second, mutations in MCa cause parallel changes in the ability of the toxin to induce LLSS on isolated channels and to modify the kinetics of ECRE. Finally, neither the wild type MCa nor its mutated analogues have any effects on the SR calcium pump (data not shown). This, on the one hand, clearly confirms that these long events are due to the calcium released through a single toxin-modified channel while calcium sparks are generated by the opening of a group of calcium release channels. On the other hand, it gives further insight into the interaction of MCa and the calcium release channel. As discussed above the toxin seems to prefer the open conformation when it binds to RyR. If MCa would only bind to the open channel the prediction would be that long events should tail calcium sparks. This is clearly the case for amphibians where long events are generally made up of a leading spark and a tailing ember. It should be noted, however, that a few events (2% of those where long embers were observed) are devoid of the leading ember. This indicates that there is a possibility, albeit much lower, for the toxin to bind to the closed channel. We, of course, cannot exclude the possibility that the binding actually occurred right after the opening of the channel and this then prevented neighboring channels to open and the spark from being formed. While the above scenario is a real, although unlikely possibility in amphibians, it clearly cannot account for the observation in mammals where all long events are without leading sparks. Taken together, our data are in line with the hypothesis that MCa and its mutants bind preferably, but not solely to the open channel.