National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 560 065, India
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Abstract |
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Keywords: cation channels/distant structural similarity/superfamily/three-dimensional modelling/transmembrane domain
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Introduction |
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Ryanodine receptor function is best understood in vertebrate skeletal muscle. It is required for the intracellular Ca2+ release that occurs prior to muscle contraction, in response to nerve impulses delivered to the muscle plasma membrane (Caterall, 1991). The other two ryR isoforms are often referred to as the `heart' and `brain' forms, but the cellular and tissue distribution of the isoforms is more complex than is suggested by this nomenclature [reviews are available (Coronado et al., 1994; Meissner, 1994
; Striggow and Ehrlich, 1996
)]. Functional studies have shown that the channel may be regulated by various endogenous effector molecules including Ca2+, ATP, cADP ribose and calmodulin, depending upon the isoforms. In addition, both Insp3R and ryR have been postulated to function during Ca2+-induced Ca2+ release in neuronal and non-neuronal tissues requiring Ca2+ oscillations (Tsein and Tsein, 1990
). The presence of these intracellular Ca2+ channels in such diverse tissues indicates that they are likely to be involved in many different cellular functions. Calcium is known to be a regulator of both the receptor channels, although no specific binding motifs are known. Both Insp3R and ryR are poorly selective and high-conductance Ca2+ channels. The estimated permeability ratio (divalent/monovalent) of both the receptors is nearly six (Tinker and Williams, 1992
; Bezprozvanny and Ehrlich, 1994
). The skeletal muscle ryR has been visualized using cryomicroscopy and angular reconstitution at 30 Å, which showed the structure of the entire system to be a mushroom-shaped tetramer with the transmembrane domain being a part of the stem (Serysheva et al., 1995
). Obtaining the detailed molecular structure of these assemblies by X-ray crystallographic or NMR techniques is challenging owing to their membrane-spanning regions and large dimensions (molecular weight 4x450 kDa).
Detailed three-dimensional structures are not available for either of these two classes of receptors. We have carried out an analysis of several Insp3R and ryR sequences with a view to identifying residues important for calcium binding. We have particularly focused on the transmembrane domain, which is involved in Ca2+ channeling. The ryR and Insp3R share sequence homology in parts (see below) and have the same quaternary structure (Wagenknecht and Radermacher, 1997). Both Insp3R and ryR occur as homotetramers where the protomers contain ~3000 and ~5000 amino acids, respectively (Mignery et al., 1989
; Serysheva et al., 1995
; Galvan et al., 1999
). It is well known that both the receptors share high sequence similarity at the C-terminal TM domain (Mignery and Sudhof, 1993
; Galvan et al., 1999
; Ramos-Franco et al., 1999
). PRODOM (Corpet et al., 1999
) records the N-terminal domain (domain id PD001922) of around 550 amino acids with ip3r_mouse-numbering 143671 and rynr_human-numbering 180650 to be similar. The N-terminal domain in the case of Insp3R was shown to be the ligand-binding domain (Mignery and Sudhof, 1990
; Miyawaki et al., 1991
). Furthermore, a middle domain of 168 amino acids (domain id PD002036; ip3r_mouse-numbering 19632131 and rynr_ human-numbering 37514123) shares high sequence similarity among Insp3Rs and ryRs. The C-terminal transmembrane domain is divided into more than one domain according to PRODOM and a region of around 300 amino acids (domain id PD001555; ip3r_mouse-numbering 23822674 and rynr_ human-numbering 46125032) shares relatively high sequence similarity (36% sequence identity). Immuno-gold electron microscopy data (Mignery et al., 1989
) and glycosylation data (Michikawa et al., 1994
) demonstrate the positions of the N- and C-termini and the location of particular loops with respect to the cytoplasm, respectively; however, knowledge of the number, location and boundaries of the TM helices is valuable.
The main objective of this paper is to discuss the similarity between the two receptors, its implications on receptor regulation by calcium and the overall structure of the channel-forming domain of the receptors. Structure prediction studies on the transmembrane region of Insp3R and ryR have been pursued which emphasize the fact that the C-terminal parts of the TM domains of the two classes of receptors, constituting the last three TM helices, share the highest similarity. This paper also reports a novel attempt at the recognition of the minimum channel requirements in Insp3R and ryRs: two transmembrane helices, the channel pore-helix and selectivity filter as observed in the potassium channels. The strong similarity between these receptors and the K+ channels has allowed the construction of a three-dimensional structural model of the C-terminal, structurally conserved helices of the transmembrane region despite differences in pore diameter and direction of ion transfer. These findings are supported by experiments using sequence analysis (Galvan et al., 1999) reported subsequent to our observations.
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Materials and methods |
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Results and discussion |
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Twelve Insp3R sequences and 13 ryR sequences were chosen and aligned at the membrane-traversing transmembrane (TM) domain. The multiple alignment of Insp3R and ryR sequences show the presence of several conserved negatively charged residues (Table I) which could act as Ca2+ binding sites. While studying Ca2+ regulation of Insp3R receptor at the molecular level and the structural determinants of Ca2+ binding, Sienaert and co-workers (Sienaert et al., 1996
, 1997
) identified eight linear sites which were shown to bind both calcium and ruthenium red (see Table I
). Of the eight sites, three are in regions where the two classes of receptors share high sequence identity. The regulatory calcium binding sites are therefore novel conserved motifs. Two EF-hand Ca2+ binding domains have been identified in lobster skeletal muscle ryR (Xiong et al., 1998
), corresponding to 40704130 of rynr_human, which are at the boundary of the middle domain conserved between Insp3R and ryR receptors. Insp3R, however, does not contain an equivalent EF-hand motif, but is replaced by an aspartateglutamate-rich region (21242146 of ip3r_mouse) which was shown to bind Ca2+ (Sienaert et al., 1997
). Conversely, a region from ip3r_mouse (amino acids 24632528), which is a segment of the C-terminal domain shown to bind Ca2+, is replaced by a highly aspartate- and glutamate-rich region in rynr_human.
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Recently, a structure of the tetrameric K+ channel (Doyle et al., 1998) from Streptomyces lividens was reported, revealing many mysteries about the channel structures that had intrigued physiologists for many decades. Apart from two membrane-spanning helices, the loop region connecting the two helices (P-loop) forms the selectivity filter. The N-terminal region of the P loop is also
-helical (which is termed pore helix), slanting towards the pore axis from outside. The helix is followed by a signature sequence: five amino acids in this zone, corresponding to VGYGD, form the lining of the selectivity filter orienting their main chain carbonyls towards the pore axis and their side chains outwards, thus stabilizing the right ions of the desired pore size. Sequence alignments from various K+ channels, both inward and outward rectifiers, show that most of the residues of the pore helix and signature sequence are conserved (Armstrong, 1998
; Doyle et al, 1998
; MacKinnon et al., 1998), suggesting that the architecture of the channels is similar, irrespective of the direction of ion transfer. Moreover, two membrane-spanning helices per monomer would be the minimum requirement and sufficient for forming the functional channel tetramer.
Prediction studies were carried out on the sequences of one Insp3R and one ryR, to map the putative transmembrane region on both the receptors. Various transmembrane region prediction methods available on SWISSPROT server (www.expasy.ch) were employed. The results from various methods with the predicted positions of the transmembrane helices are shown in Figure 1 for ip3r_mouse sequence. We confirmed this result by applying these methods to the KcsA sequence, where all methods miss the pore helix. The helix-wheel diagram is shown in Figure 2
for the region predicted to contain the sixth TM helix of ip3r_mouse by PERSCAN (Donnelly et al., 1994
). It is clear from the prediction studies reported here that Insp3R contains a topology of six membrane-spanning helices.
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Figure 3 shows the multiple sequence alignment of the region containing the putative last three helices of both the receptors where the highest sequence similarity extends to a further 100 amino acids towards the C-terminus (36% sequence identity). The predicted helix positions and certain conserved amino acid positions are indicated. This is also in agreement with deletion studies on Insp3R which demonstrate that the deletion of the first four TM helices of recombinant Insp3R forms functional calcium channels and mutants lacking the last two helices do not form detectable channels (Ramos-Franco et al., 1999
).
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From the above discussion and the sequence alignment shown in Figure 3, it is clear that the conserved C-terminal region also contains the predicted pore helix, which has a length of 10 amino acid residues. Following the pore helix, a motif, GXRXGGGXGD (starting from 4820 of ryRs and 2540 of Insp3Rs), is found to be highly conserved in all known Insp3Rs and ryRs. Mutation of glycine to alanine in this signature sequence in ryR, at the first, fourth and sixth positions, disrupts the calcium release from the channel (Zhao et al., 1999
). Also, the isoleucine to threonine mutation of ryR-1 (see Figure 3
) decreases the threshold of Ca2+ required to initiate opening of the wild-type channel and results in a reduced release of Ca2+ from internal stores (Balshaw et al., 1999
; Lynch et al., 1999
). These data suggest that this conserved region constitutes channel conduction pathway or the central pore lining of this receptor (Zhao et al., 1999
), reaffirming that the same topology is present in the channel-forming region as in the KcsA K+ channel, viz. fifth helix, pore helix, pore-lining region and sixth helix.
It is anticipated that Ca2+ channels have pores that are related architecturally to K+ channels (Doyle et al., 1998; Roux and MacKinnon 1999
). In this paper, we report the three-dimensional structure of ryR human TM domain using the KcsA structure as the template and by employing the COMPOSER homology modeling program (Sutcliffe et al., 1987
; Blundell et al., 1988
; Srinivasan and Blundell, 1993
). The transmembrane helices, pore helix and selectivity filter region are considered as structurally conserved regions (SCRs) and the resulting structure is energy minimized with a fixed backbone conformation. The tetramer positions of the calcium channel are generated from the K+ channel tetramer by a structure superposition program called SUPER (B.S.Neela, personal communication). Figure 4a
shows a ribbon diagram of the tetramer model of ryR human derived by such comparative modeling studies. The presence of leucines and other hydrophobic residues in two adjacent protomers at their interface (see Figure 4a
) might account for the stability of the tetramer.
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Conclusions
We have identified three regions of Insp3R and ryR which contain high similarity and are important for Ca2+ binding and channel regulation. The high degree of partial sequence similarity between the two receptors suggests that the elements involved in calcium channel formation and selectivity are highly similar and conserved during evolution. It is well known that all of the known Na+, Ca2+ and K+ channels are made of tetramers of either four internal repeats each containing six membrane spanning helices or four protomers each having six membrane-spanning helices (Hille, 1992). Some channels are tetramers of two transmembrane-spanning
-helices. On the basis of structural principles exemplified by the KcsA K+ channel structure (Doyle et al., 1998
), we have put forth the first atomic level structure of a calcium channel, a single file pore, which is in agreement with existing structural and theoretical studies, which provides clues to the permeation pathway located in the linear sequence and how calcium ions might pass through it. The coordinates of the tetramer model are available from the authors on request. To our knowledge, no previous papers have mentioned a pore helix in Insp3R or ryRs. The above analysis also confirms that the cationic channel proteins belong to a broad superfamily. It will be interesting to compare the four internal repeats of the Na+ channels for similarities in secondary structural features.
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Note added in proof |
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Notes |
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Acknowledgments |
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References |
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Received July 12, 2000; revised December 5, 2000; accepted June 18, 2001.