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Chapter 5 THE PORE OF THE RYANODINE RECEPTOR

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(1)

THE PORE OF THE RYANODINE RECEPTOR CHANNEL

Alan J. Williams

1

, S.R. Wayne Chen

2

, and William Welch

3

1

Cardiac Medicine, National Heart & Lung Institute, Imperial College London;

2

Cardiovascular Research Group, Dept. of Physiology and Biophysics, and Dept. of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada;

3

Dept. of Biochemistry, University of Nevada School of Medicine, Reno, NV

THE PHYSIOLOGICAL ROLE OF THE RYANODINE RECEPTOR CHANNEL

As is highlighted in various other chapters of this book, the ryanodine receptor functions as an intracellular membrane channel;

providing a regulated pathway for the movement of from a storage organelle, such as the sarcoplasmic reticulum in muscle, down an electrochemical gradient to initiate a wide variety of cellular processes.

96

The efficiency of the ryanodine receptor as a channel in a process such as excitation-contraction coupling is underpinned by both its ability to open and close in response to appropriate stimuli and its ability to allow the movement of very large numbers of ions per unit time. This second component of RyR channel function reflects both the capability of the channel to discriminate between ions present in the lumen of the sarcoplasmic reticulum and to maximize rates of ion throughput.

In this chapter we will discuss the mechanisms and structural features of

the pore region employed by the RyR channel to select and then to

translocate across the sarco(endo)plasmic reticulum membrane. We

will also consider the emerging proposition that the RyR pore contains a

high-affinity binding site for ryanodine.

(2)

44

WHAT AND WHERE IS THE RYR PORE?

An individual RyR channel is composed of four identical monomers, each of which contains approximately 5000 amino acid residues (variations between isoforms are described in Williams et al.

147

). It has been proposed that each RyR monomer could contain a viable pore and that the function of an individual RyR channel reflects coordinated gating of these four pores.

148

However, a growing body of evidence, arising from the investigation of the function of heteromeric channels containing wild type and mutant monomers,

37

and studies of the influence of chemical modifiers of channel function,

149

indicates that each tetrameric RyR channel contains a single pore.

The pore of any membrane ion channel is the region of the molecule that provides a pathway for the movement of ions across the dielectric barrier of a phospholipid membrane. As a consequence, this region of the RyR channel will inevitably be formed by membrane spanning regions of the molecule.

Potential membrane-spanning regions of the RyR monomer have been identified in the C-terminal 1000 residues with the number of transmembrane sequences present in each monomer estimated at between 4

3

and 10.

7,8

Experiments involving tryptic cleavage

150

or truncation of RyR channels

29,73

indicate that the region of the molecule encompassing the probable transmembrane domains can, in isolation, provide a pathway for cation translocation.

Balshaw et al.

77

have proposed a role for a specific sequence of residues in the formation of the RyR pore. These authors noted that a mutation in the human RyR1 that underlies an unusually severe form of central core disease (I4898T)

151

occurred in a sequence of residues that shared a degree of homology with the consensus selectivity sequence of channels (Fig. 5-1).

Balshaw et al.

77

also noted that this sequence of residues was found in a loop, equivalent to the pore forming or P-loop in channels, which links the probable penultimate and last transmembrane domains of all topology models of RyR. This potential RyR P-loop occurs within the lumen of the SR and Balshaw et al. proposed that, as is the case for channels, the loop could fold back into the membrane to contribute to the formation of the RyR pore.

Evidence in support of the involvement of residues, analogous to the

channel selectivity filter and within a RyR P-loop, in ion translocation has

been provided by investigations of the properties of individual channels in

which specific residues have been mutated; an approach that has proved to

be invaluable in the identification of selectivity sequences in other ion

channels. Residue substitutions in and around the putative selectivity filter

(3)

sequences in both RyR1

38

and RyR2

37,74

decrease, to a greater or lesser extent, unitary conductance of both and

Figure 5-1. Signature selectivity sequences of representative channels (bold) and analogous motifs of three rabbit RyR isoforms are shown in A. In channels these motifs occur in an extracellular pore forming or P loop linking S5 and S6 in voltage-activated channels (as shown in B) and M1 and M2 in KcsA. The putative RyR selectivity sequence occurs in an analogous position in this species of channel between the penultimate and last transmembrane segments (M8 and M10 in the topology model described in Du et al.

8

and shown in C). In both B. and C. the P-loop (bold) is shown folding back into the membrane to contribute to the formation of the pore.

Analogies between the pore regions of RyR and K channels

The foregoing discussion indicates certain analogies between the pore

regions of RyR and channels. Both species of channel are

homotetramers; in both cases the pore of the channel appears to be formed

by elements of an equivalent extracellular/luminal loop linking the last two

transmembrane domains and within this loop RyR contains a sequence of

residues analogous to the selectivity sequence of the channels. Whilst we

have no direct information concerning the structure of the RyR pore, a

wealth of structural information has been made available for the pore of

(4)

channels as the result of the crystallization of simple bacterial channels.

9,152

The essential features of these structures are as follows. The pore is formed at the long axis of four identical subunits. Each subunit contributes two transmembrane helices (in KcsA the outer helix (M1) and the inner helix (M2)). The extracellular loop linking these helices folds into the membrane and itself contains two elements of differing secondary structure, a short helix referred to as the pore helix and an extended chain of residues including the identified selectivity sequence. When the channel is closed, a gate is formed at the crossing of the four inner helices at the cytosolic entrance to the structure.

9

The transition to an open conformation involves a flexing of the inner helices at a glycine hinge located somewhere near the middle of each helix.

153

A ion entering the pore from the bulk solution at the cytosolic side of the open channel is stabilized in a water- filled cavity by dipoles of the four pore helices.

9

On entering the selectivity filter of the channel the is dehydrated and coordinated by backbone carbonyl oxygens of the signature selectivity residues.

154,155

The cation is re- hydrated as it leaves the filter and re-enters the extracellular bulk solution.

155

The extraordinary powers of discrimination of channels arise from the very precise coordination of this ion within the fairly rigid selectivity filter;

ions are not coordinated and are hence excluded, as the filter is a poor solvent for relative to water. In these channels, rates of translocation are maximized by electrostatic repulsion between ions occupying multiple sites within the selectivity filter.

Examinations of predictions of the secondary structure of elements of the putative RyR P-loop have identified a helical region in the RyR P-loop that occurs in a location equivalent to the pore helix in channels (Fig. 5- 2).

147,156

The sequence of structural elements in KcsA is M1 (outer helix);

pore helix; selectivity filter; M2 (inner helix). In RyR the elements occur in an equivalent order (using the topology profile recently described by Du et al.

8

) M8 (transmembrane helix); M9 (putative pore helix); loop containing residues analogous to channel selectivity sequence; M10 (transmembrane helix). These observations have led to the suggestion that the putative RyR P-loop may adopt a tertiary configuration equivalent to that determined for the P-loop of KcsA and that the pore of the RyR channel could, as a consequence, adopt a broadly similar structure to that determined for KcsA (Fig. 5-3).

147,156

In such an arrangement an ion entering the pore from the SR luminal

bulk solution would encounter a region formed by the apposition of the four

chains containing residues analogous with the channel selectivity

sequence. Having passed through this region the ion would enter a water-

filled cavity lined with residues of the M10 helices before leaving the pore

into the cytosolic bulk solution. A general structure of this form would

(5)

provide the RyR pore with a means to accomplish the fundamental requirement of a membrane ion channel. As in KcsA the presence of a water-filled cytosolic cavity, into which are orientated four helix dipoles, would effectively bring the cytosolic bulk solution into the membrane and provide a means of overcoming the potential electrostatic destabilisation of a cation within the membrane.

Figure 5-2. Pore forming loops of KcsA and RyR. A . The P loop of KcsA contains a short helical element referred to as the pore helix (pore) and a signature selectivity sequence (S).

Secondary structure predictions for the putative RyR P loop have identified motifs equivalent to the channel pore helix in all isoforms of the channel. The residues contributing to the putative pore helices of three rabbit isoforms of RyR and their location within the P loop are shown in panel B. In both panels residues of the selectivity sequence are underlined.

Evidence in support of this putative structure

Whilst there are very significant fundamental differences in the characteristics of ion discrimination and translocation in RyR and channels (see later), there are some similarities that could support the suggestion that the channels share a basically similar pore structure.

Permeant cation movement is blocked in both species of channel by a range

of tetra alkyl ammonium cations.

147

The demonstration of block of RyR by

large polycations

157

and channel N-type inactivation peptides from the

bulk solution at the cytosolic face of RyR

158

would be consistent with the

(6)

48

proposal that access to the site of block in a putative selectivity filter is via a large water-filled cavity at this entrance to the structure.

We have already indicated that residue substitution in the selectivity filter of the putative RyR pore modifies rates of monovalent and divalent cation translocation. It should also be noted that expression of heteromeric mouse RyR2 channels comprising different combinations of wild type monomers and monomers in which an alanine residue has been substituted for glycine 4824 (a substitution that reduces conductance to 3% of wild type conductance when expressed as a homotetramer) gives rise to channels with a range of conductance values intermediate between wild type and homomeric G4824A.

37

These observations indicate that each monomer contributes to ion handling and would suggest that a single pore is formed at the axis of the tetramer.

Figure 5-3. The KcsA pore (pdb 1BL8 from Doyle et al.

9

) is shown in the left-hand panel;

two diagonally apposed monomers have been removed for clarity. The two transmembrane helices of each monomer are shown in grey, the pore helices in black and the residues of the selectivity filter in ball and stick representation. The equivalent arrangement of structural elements identified in the RyR putative P loop, are shown in the right-hand panel.

Further support for this putative pore structure comes from the recent

identification of residues of RyR1 that are involved in interactions with the

sarcoplasmic reticulum luminal accessory protein, triadin. Lee et al.

159

identified three acidic residues in the luminal loop linking M8 and M10 that

appear to be critical for this interaction. As noted by these authors all three

residues would be located at the luminal face of the putative RyR pore

described in this chapter, with one located towards the N-terminus of the

(7)

putative pore helix and two others in the loop containing the selectivity sequence residues linking the putative pore helix and M10.

Finally, models of the proposed pore region of RyR, incorporating a putative pore helix, have been produced by extrapolating from the crystal structure of KcsA.

156,160

ION SELECTION AND TRANSLOCATION IN RYR

The various observations outlined above have led us to conclude that it is reasonable to hypothesize that the pore of the RyR channel shares a broadly common architecture with the equivalent region of channels. However, a comparison of the discrimination and translocation properties of these two species of channel suggests that the mechanisms underlying these processes must be very different. channel function is characterized by high rates of translocation coupled with extremely high specificity and these intuitively contradictory properties are made possible by a pore and selectivity filter structure that permits very precise coordination of and electrostatic interactions between adjacent cations in the filter.

161

The ion handling properties of RyR have been reviewed in detail in Williams et al.

147

and are summarized here. The first observation to emerge from these investigations is that RyR is not a particularly selective channel.

While it does completely exclude anions, RyR discriminates only relatively poorly between cations. Unlike channels, RyR is permeable to a very wide range of divalent and monovalent inorganic cations and some organic monovalent cations. Calculations of relative permeability from reversal potentials monitored for single RyR channels under bi-ionic conditions reveal that, as a group, the alkaline earth divalents are essentially equally permeant in RyR. Similarly, RyR is equally permeable to the group 1a monovalent cations. However RyR is able to discriminate to some extent between divalent and monovalent inorganic cations with divalents 6 to 7 fold more permeant than monovalents.

Another striking feature to emerge from these studies is the rate of both

monovalent and divalent cation translocation achieved in RyR. Unitary

conductance for increases with increasing activity and plateaus at

approximately 1nS. Equivalent experiments with demonstrate a

maximal unitary conductance of approximately 200pS. Despite these very

high rates of cation translocation and very limited discrimination, permeant

cations interact with the RyR pore with high affinity. Values obtained from

conductance-activity relationships and from rate theory modelling indicate

that 50% maximal conductance is achieved at activities of for

alkaline earth divalents and activities of 10-20 mM for group 1a

(8)

monovalents. While rates of cation translocation in RyR are significantly greater than those in channels all available evidence indicates that this does not reflect multi-ion occupancy in a RyR selectivity filter; rather RyR behaves as a single-ion channel. Theoretical considerations indicate that to achieve the measured rates of cation translocation with single ion occupancy, RyR is likely to possess a short, wide, selectivity filter. In agreement with this suggestion, investigations involving a range of permeant and impermeant monovalent and divalent organic cations have produced estimates of approximately 3.5 Å for the minimum pore radius and approximately 10 Å for the length of the voltage drop across the channel.

Even this very brief comparison of the ion handling properties of RyR and channels indicates that the mechanisms underlying discrimination and translocation are likely to be very different in the two channels. If, as we have proposed, the channels share a broadly comparable pore structure to overcome the dielectric barrier of the membrane, it is clear that the arrangement of structural elements and hence the processes governing interactions of cations with the pore must be very different in the two structures.

DOES THE RYR PORE CONTAIN A RYANODINE- BINDING SITE?

The RyR channel is so named because each functional channel contains a high-affinity binding site for the plant alkaloid ryanodine and, as described elsewhere in this book, the binding of ryanodine to this site has dramatic consequences for both channel gating and rates of cation translocation.

Initial attempts to identify the ryanodine binding site within RyR1 involved proteolysis and photoaffinity labelling.

82,162

These approaches localised the site to a 76 kD portion at the C-terminus of the molecule which encompasses the putative pore region discussed in this chapter. The suggestion that a ryanodine- binding site is located within the pore of RyR has arisen as the result of several observations. It is well established that high affinity ryanodine binding involves interaction of the ligand with an open conformation of the channel.

163,164

This could mean that the binding site is located within the pore and is only accessible when the channel is open or that a site outside the pore is made available by a conformational alteration associated with channel opening.

Single channel investigations of the interactions of derivatives of ryanodine

(ryanoids) have demonstrated that these events are influenced by

transmembrane holding potential

164,165

and, whilst it has been established that

the vast majority of this voltage dependence arises from a potential-driven

alteration in receptor affinity,

166

the possibility remains that ryanoids might

(9)

bind at a site at the extremity of the voltage drop across the channel; such a site is likely to be within the pore.

Also contributing to a proposed pore location for a ryanodine-binding site is the observation that the mutation of several residues located within the putative RyR P-loop alters the interaction of ryanodine with the channel. Substitution of residues in and around those equivalent to the channel selectivity sequence has been shown to prevent the binding of to populations of channels or to either increase or decrease the affinity of the interaction.

37,38,74,167

Investigations in which the interactions of charged ryanoids with individual RyR channels have been monitored have revealed that the high- affinity ryanodine binding site is only accessible from the cytosolic side of the channel.

164

We have investigated the possible involvement of the putative cytosolic cavity in ryanodine binding by investigating the interactions of the ligand with mouse RyR2 channels in which residues of M10, the helix that lines the cavity of the proposed RyR pore, have been mutated. Alanine substitution of several of these residues results in alterations in the response of channels to and caffeine

78

and reduced levels of binding.

79

One substitution has been studied in detail by monitoring ryanodine interactions with individual, voltage- clamped, channels. Although we were unable to detect binding of

to populations of Q4863A channels, single channel investigations revealed that ryanodine does interact with the channel. On the timescale of a single channel experiment the interaction of ryanodine with wild type RyR channels is irreversible; observations with Q4863A revealed that this mutation produced a dramatic increase in the rate of dissociation of bound ryanodine from the channel so that, in the continued presence of ryanodine, we observed reversible interactions of the ligand with the channel.

Interestingly the consequences of the Q4863A substitution appear to be very specific.

79

These channels respond to physiological and pharmacological regulators of gating in a manner equivalent to wild type channels. Similarly, unitary conductance and the fractional conductance of the Q4863A RyR- ryanodine complex have the same values as these parameters in wild type channels. Finally, rates of ryanodine association with, and dissociation from, Q4863A channels are sensitive to changes in transmembrane holding potential and these parameters vary in a manner analogous to those of reversible ryanoids such as ryanodol and

with wild-type sheep RyR2 channels. Taken together, the effects of residue

substitution in both the putative selectivity filter and M10 indicate that

residues of the proposed pore are likely to make an important contribution to

the high affinity binding site for ryanodine in RyR.

(10)

CONCLUDING REMARKS

In this chapter we have given a very brief overview of the evidence for the involvement of residues of a C-terminal luminal loop in the formation and function of the RyR channel pore. Further investigations will be aimed at elucidating the relationships between pore structure and the mechanisms that a) give rise to the very unusual characteristics of ion discrimination and translocation displayed by RyR, and b) contribute to the binding of ryanodine to the channel.

ACKNOWLEDGMENTS

The work in the authors’ laboratories is supported by research grants from

the British Heart Foundation to A.J.W., the Canadian Institutes of Health

Research and the Heart and Stroke Foundation of Alberta, Northwestern

Territories, and Nunavut to S.R.W.C, and the National Science Foundation

(MCB9817605) to W.W.

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