EVOLUTION OF THE RYANODINE RECEPTOR GENE FAMILY
, A.K.M.M. Mollah1
, and Xander H.T. Wehrens2
1Dept. of Biology, Yeshiva College, New York, NY; 2Dept. of Physiology and Cellular Biophysics, Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia
University, New York, NY
Fluctuations in cytosolic calcium concentrations act to modulate a vast array of second messenger signaling pathways in living organisms. This control mechanism results in delicate regulation due to low cytosolic levels under resting conditions alternating with rapid and transient increases in upon stimulation. Key components of this pathway were initially evolved in primordial transport systems and then rapidly developed into more specialized pumps and channels on the plasma membrane.
Following the evolution of the sarcoplasmic reticulum (or in some organ systems endoplasmic reticulum) as intracellular stores, intracellular calcium-release channels evolved.
Intracellular calcium-release channels are required for many cellular
processes, such as excitation-contraction coupling in skeletal and cardiac
muscle and signal transduction in the nervous system.1
The system of
endomembranes that forms the sarco(endo)plasmic reticulum plays a vital
role in handling in most eukaryots.2
Two families of intracellular
calcium-release channels are found in this compartment: ryanodine receptors
(RyRs) and inositol 1,4,5-trisphosphate receptors The RyRs (560
kDa) and (260 kDa) are similar in structure, both having large N-
terminal and small C-terminal domains protruding into the cytoplasm, and
several transmembrane regions near the C-terminus (Fig. 1-1).3,4
Ryanodine receptors consist of four monomers that assemble to form a functional calcium-release channel. The large N-terminal domain contains binding sites for channel modulators that regulate the channel pore including FK506-binding proteins FKBP12 (calstabin1) and FKBP12.6 (calstabin2), which are bound to RyR1 and RyR2, respectively (Fig. 1-1).5
FKBP12/12.6 binding to RyR stabilizes the channel in the closed-state confirmation. Three leucine/ isoleucine zipper (LIZ) motifs on RyR2 allow binding of the adaptor proteins spinophilin, PR130, and mAKAP, which target the protein phosphatases PP1 and PP2A, and protein kinase A (PKA) to the channel complex, respectively (see Chapter 15).6
In the original topological model for RyR1 proposed by Numa and colleagues,3
four transmembrane (TM) segments were proposed near the C- terminus. Subsequently, Zorzato et al.7
proposed 12 hydrophobic sequences in the C-terminal region of RyR (see Chapter 2 for a more extensive review).
The first two TM sequences (M’ and M”) were considered to be very tentative, and the others were called M1-M10.7
Based on an experimental study, MacLennan et al.8
proposed a slightly different model (Fig. 1.1), in which the C-terminus contains six to eight TM segments. In addition, the pore segment (M9) is thought to line the pore of the channel as a selectivity filter described for voltage-gated ion channels,9
allowing ions to transverse the membrane. This chapter will focus on the genetic characteristics and phylogenetic relationships among different RyR isoforms that have been identified thus far.
Figure 1-1. Schematic diagram of the RyR protein. A. Line diagram showing the RyR protein with the transmembrane (TM) domains and pore. Also shown are the leucine/isoleucine zippers (LIZ) and binding-site of FKBP12/FKBP12.6. B. Proposed organization of the transmembrane domains, according to MacLennan et al.8 TM domains are numbered according to the Zorzato model.7
RYANODINE RECEPTOR GENES
Ryanodine receptors were first cloned from mammalian skeletal and cardiac muscle.3,4,10
Analysis of the nucleotide sequences revealed that these two subtypes are about 66% homologous.3,4
A third mammalian RyR isoform was cloned from rabbit brain and a mink lung epithelial cell line.11,12
Similar RyR isoforms have been cloned from non-mammalian vertebrates (e.g., chicken, bullfrog, and blue marlin),13-15
although they seem to express only two distinct isoforms. The non-mammalian vertebrate isoforms appear to be homologs of the mammalian skeletal muscle isoform RyR1, while the isoforms are related to the mammalian RyR3.13,15
Although RyRs were originally discovered in vertebrates, more recently they have been identified in invertebrates, including Caenorhabditis elegans and Drosophila melanogaster (Table 1-1).16,17
The gene for RyR1 is located on chromosome 19q13.2 in humans, and spans 104 exons. The RyR2 gene with 102 exons is located on chromosome 1q43, and the RyR3 gene with 103 exons on chromosome 15q13.3-14.
Although the RyR genes are located on different chromosomes in mice
(chromosomes 7A3, 13A2, and 2E4, for RyR1, RyR2, and RyR3,
the neighboring genes on these chromosomes are similar to those found on the corresponding human chromosomes. The organization in the phylogenetic tree supports the model that an expansion of the vertebrate gene family was associated with an initial duplication of the RyR1 gene, since only one RyR gene is found in invertebrates (Fig. 1-2). Indeed, non- mammalian vertebrates express two RyR genes.15
It appears that a second duplication occurred in mammals, which distinguishes them from other vertebrates. This model is consistent with the theory that gene duplication is important in adaptive evolution because it allows the new proteins to have distinct functional characteristics.26
An additional means of generating diversity in RyR channels is through alternative splicing. Alternative splicing has been demonstrated for several vertebrate18,27,28
and invertebrate RyR isoforms.29
Although some data suggest that alternatively spliced variants of RyR3 exhibit reduced caffeine sensitivities, relatively little is known about the electrophysiological properties of alternatively spliced RyR channels.30
Considering that invertebrate species appear to have only 1 RyR gene, in contrast to the three mammalian RyR genes, it is likely that invertebrates and vertebrates use fundamentally different means of generating diversity in RyR function.
Indeed, there is evidence for alternative splicing of the insect H. virescens RyR gene, resulting in potentially different channels.31
Thus, the major means of generating diversity in invertebrate RyR channels may involve alternative splicing, whereas vertebrate RyR diversity may result primarily from the presence of multiple genes.
PHYLOGENY OF RYANODINE RECEPTORS
Based on phylogenetic analysis, it is likely that all RyR isoforms evolved from a single ancestor (Fig. 1-2), and possibly that RyR and evolved from a common ancestral channel.32
It is thought that have 6 and RyR have 6-8 transmembrane domains, although definitive structural evidence is currently lacking.8,33
Sequence homology comparison has revealed a high degree of homology between the pore region and TM domains lining the pore region comparing RyRs and32
Ryanodine receptors have not been detected in protozoa or algae. Although there seems to be some functional evidence for calcium-release channels in higher plants, primordial RyR seems to occur in C. elegans.34
No data are available yet on the origin and time of appearance of
intracellular calcium-release channels.35
However, an overall structural
homology is present between the pore-forming region of ryanodine receptors
and the superfamily of ion channels that encompasses most of the voltage-
5 gated ion channels, the cyclic-nucleotide-gated channels and the transient receptor potential (TRP) channels.36
There is ~35-40% homology at the amino acid level between C. elegans and vertebrates. The homology is higher in certain functionally important domains that are conserved among all RyR isoforms, including the leucine/isoleucine zipper domains, the pore region, and the transmembrane domains. The most similarity between the different isoforms is observed in the pore region, and the adjacent two TM regions (M8 and M10), in which many residues are conserved from C.
elegans to the human RyR isoforms (Fig. 1-3). Mutations of many of the highly conserved amino acids in the ion-conducting pore region have diverse effects on channel functions such as caffeine-induced release, ryanodine binding, single channel conductance and modulation, and cation selectivity.37,38
Figure 1-2. Phylogenetic tree of the RyR gene family. RyR protein sequences were aligned using ClustalW, and gaps were removed according to the method of Chiu et al.39 Shown is the most-parsimonious tree using PAUP software. Numbers at each node represent bootstrap values (the probabilities that two lineages are joined together at the node to form a single cluster). Lengths of horizontal lines of the tree are proportional to the estimated number of amino acids substitutions. Abbreviations for the RyR isoforms are described in Table 1-1.
DIFFERENTIAL EXPRESSION OF RYR ISOFORMS
Expression of RyR1 is relatively abundant in skeletal muscle, although it is also expressed at lower levels in cardiac and smooth muscle, cerebellum, testis, adrenal gland, and ovaries.3,10,13,40,41
Whereas RyR1 is predominantly expressed in Purkinje cells in the brain, RyR2 is localized mainly in the somata of most neurons.40
RyR2 is expressed robustly in the heart and brain, and at lower levels in the stomach, lung, thymus, adrenal gland, and ovaries.27,41
The RyR3 isoform is expressed in the brain, diaphragm, slow twitch skeletal muscle, as well as several abdominal organs.11-13,41
The non- mammalian isoform is expressed strongly in skeletal muscle and weakly in brain, whereas the isoform is expressed in a variety of tissues, including skeletal and cardiac muscle, lung, stomach, and brain.15
There is some evidence that alternative splicing of RyR genes may underlie the tissue-specific expression of certain isoforms.28
Figure 1-3. The ryanodine receptor pore-forming region. A. Alignment of 17 RyR isoforms showing the pore region and transmembrane regions 8 and 10 (M8, M10). Numbers 1 and 2 indicate regions not shown in alignment. B. The bar graph indicates the number of different amino acids among the different isoforms at the indicated residues.