CLIC2-RyR1 Interaction and Structural Characterization by Cryo-electron Microscopy

https://doi.org/10.1016/j.jmb.2009.01.059Get rights and content

Abstract

Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.

Introduction

The chloride ion is the most abundant anion in the tissues of animals and plants, and anion channels in the cells are often referred to as chloride channels. Several classes of chloride channels have been found, of which three are well-characterized: the ligand-gated receptors, the cystic fibrosis transmembrane conductance regulators (CFTR), and the chloride ion channels (CLC).1 Chloride ion channels are involved in regulation of absorption and secretion of Na+, setting the cell membrane potential, acidification of cytoplasmic organelles, and regulation of cell volume.2

As a new class of chloride channels, chloride intracellular channel (CLIC) proteins, differ from the other classes of chloride ion channels in primary structure and in the transmembrane regions of the tertiary structure.3 Since the first member of CLIC, p64 (CLIC5), was discovered in bovine kidney, several members of the CLIC family have been found in other tissues from many species, including NCC27 (CLIC1), CLIC2, CLIC3, mtCLIC (CLIC4), and parchorin (CLIC6).4, 5, 6, 7, 8, 9, 10, 11 With the exception of p64 and parchorin, these proteins are composed of ∼ 240 amino acid residues. The CLIC proteins show sequence homology with members of the glutathione-S-transferase (GST) superfamily.12 Another feature of CLIC proteins distinguishable from other ion channels is that they exist in two different forms: either as soluble globular proteins, or as an integral membrane protein that is incorporated into lipid bilayers and forms ion channels.7, 1013, 14, 15, 16, 17, 18, 19

Human CLIC2 protein is composed of 247 amino acid residues and is found in many organs, including the spleen, lung, liver, and in both skeletal and cardiac muscles.6, 20 Consistent with their high degree of primary structure homology, CLIC2 is similar to CLIC1 and CLIC4 in terms of tertiary structure.17,21, 22, 23 Like other members of the CLIC family, CLIC2 can exist as a soluble globular protein, or incorporate into a lipid bilayer to form a Cl channel.17

While the membrane-incorporated CLIC2 proteins function as Cl channels, the physiological function of the soluble form CLIC proteins is less well defined. Recently, Dulhunty et al showed that CLIC2 can interact with the cardiac ryanodine receptor (RyR2) and modulate its calcium release channel activity, implying that CLIC proteins may play a role in the regulation of Ca2+ signaling.20, 24 Ryanodine receptors are the major Ca2+ release channels in both cardiac and skeletal muscle, and they play a crucial role in the Ca2+ signaling pathway that governs the muscle excitation–contraction coupling.25 The gating of RyRs is regulated with a various intracellular messengers, including calmodulin, FK-binding protein, ATP, Ca2+, Mg2+, and protein kinase A.26 The investigations reported by Dulhunty et al raise several issues: (1) can CLIC2 act as a modulator of the skeletal ryanodine receptor channel (RyR1) and, if so, is CLIC2 a common regulator of both RyR1 and RyR2? (2) Does CLIC2 modulate RyR1 differently from RyR2? (3) How does CLIC2 interact with RyR and regulate its channel activity?

In this study, we attempted to answer the above questions by employing several biochemical and electrophysiological approaches, including [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, and single channel recording; and ultimately by using the structural approach of 3D cryo-electron microscopy (cryo-EM) and single-particle image processing. Taken together, our results provide for the first time a direct evidence for a physical interaction of CLIC2 with RyR1, and they suggest that the interaction between CLIC2 and RyR1 stabilizes the closed state of the Ca2+ release channel.

Section snippets

CLIC2 facilitated [3H]ryanodine binding to skeletal heavy SR and purified RyR1

Ryanodine is a plant alkaloid that binds to the open state RyR/Ca2+ release channel with high affinity.27 To test whether CLIC2 can interact with RyR1 and modify its function, we first carried out [3H]ryanodine binding experiments. As shown in Fig. 1a, at a [Ca2+] of 10 μM, CLIC2 increased [3H]ryanodine binding to skeletal heavy SR; binding rose from 1.31 ± 0.1 pmol/mg (buffer, n = 6) to 1.57 ± 0.03 pmol/mg (15 μM CLIC2, n = 6) and then to 1.7 ± 0.01 pmol/mg (30 μM CLIC2, n = 6).

To assess whether this

Discussion

CLIC proteins are found in both skeletal and cardiac muscle of humans and other vertebrates.6, 20 A feature of CLIC proteins that is distinctive from other chloride channels is that they exist in two different forms: soluble and membrane-bound. While the membrane-bound CLIC proteins function as Cl channels, the function of soluble CLIC proteins is not known. Dulhunty et al. showed recently that CLIC2 could interact with RyR2 and modulate its channel activity, demonstrating that CLIC2 is a RyR2

Chemicals

All chemicals were of analytical grade or above and were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) unless specified otherwise.

CLIC2 expression and purification

CLIC2 was expressed and purified as described.23 Briefly, CLIC2 was expressed with an N-terminal His6 tag and purified by Ni2+-chelating column chromatography and gel-filtration chromatography. The final buffer in which purified CLIC2 was dissolved was 20 mM Tris–HCl (pH 7.5), 200 mM NaCl.

Preparation of skeletal heavy SR vesicles

Skeletal heavy SR (heavy SR) vesicles were prepared from New Zealand

Acknowledgements

This work was supported by research grants from the Natural Science Foundation of China, the Trans-Century Talent Awarding Program, Ministry of Education, and the National Basic Research Program (973 Program), Ministry of Science and Technology, P. R. China to C.C.Y., research grants from the British Heart Foundation to A.J.W., by American Heart Association grant 0430076N to Z.L., and by National Institutes of Health grant AR40615 to T.W. We thank Bin Geng (Department of Physiology, The Health

References (63)

  • ProutskiI. et al.

    Overexpressed chloride intracellular channel protein CLIC4 (p64H1) is an essential component of novel plasma membrane anion channels

    Biochem. Biophys. Res. Commun.

    (2002)
  • BerrymanM. et al.

    CLIC-5A functions as a chloride channel in vitro and associates with the cortical actin cytoskeleton in vitro and in vivo

    J. Biol. Chem.

    (2004)
  • BoardP.G. et al.

    CLIC-2 modulates cardiac ryanodine receptor Ca2+ release channels

    Int. J. Biochem. Cell Biol.

    (2004)
  • HarropS.J. et al.

    Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-Å resolution

    J. Biol. Chem.

    (2001)
  • MeissnerG.

    Molecular regulation of cardiac ryanodine receptor ion channel

    Cell Calcium

    (2004)
  • SharmaM.R. et al.

    Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor

    J. Biol. Chem.

    (1998)
  • SharmaM.R. et al.

    Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy

    J. Biol. Chem.

    (2000)
  • WagenknechtT. et al.

    Direct localization of the tRNA–anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging

    J. Mol. Biol.

    (1988)
  • SeryshevaI.I. et al.

    Structure of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP

    Biophys. J.

    (1999)
  • LiuZ. et al.

    Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel

    J. Mol. Biol.

    (2004)
  • MengX. et al.

    Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

    J. Biol. Chem.

    (2007)
  • LiuZ. et al.

    Localization of a disease-associated mutation site in the three-dimensional structure of the cardiac muscle ryanodine receptor

    J. Biol. Chem.

    (2005)
  • PerezC.F. et al.

    Amino acids 1-1,680 of ryanodine receptor type 1 hold critical determinants of skeletal type for excitation-contraction coupling. Role of divergence domain D2

    J. Biol. Chem.

    (2003)
  • WangR. et al.

    Localization of an NH2-terminal disease-causing mutation hotspot to the “clamp” region in the three-dimensional structure of the cardiac ryanodine receptor

    J. Biol. Chem.

    (2007)
  • ZhouQ. et al.

    Structural and functional characterization of ryanodine receptor-natrin toxin interaction

    Biophys J.

    (2008)
  • BrillantesA.B. et al.

    Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein

    Cell

    (1994)
  • GaburjakovaM. et al.

    FKBP12 binding modulates ryanodine receptor channel gating

    J. Biol. Chem.

    (2001)
  • SamsóM. et al.

    Structural characterization of the RyR1-FKBP12 interaction

    J. Mol. Biol.

    (2006)
  • SeryshevaI.I. et al.

    Structure of Ca2+ release channel at 14 Å resolution

    J. Mol. Biol.

    (2005)
  • ChuA. et al.

    Isolation of sarcoplasmic reticulum fractions referable to longitudinal tubules and junctional terminal cisternae from rabbit skeletal muscle

    Methods Enzymol.

    (1988)
  • InuiM. et al.

    Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle

    J. Biol. Chem.

    (1987)
  • Cited by (43)

    • Interaction of the Homer1 EVH1 domain and skeletal muscle ryanodine receptor

      2019, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      All chemicals, unless otherwise specified, were purchased from Sigma-Aldrich. Skeletal sarcoplasmic reticulum (SR) vesicles were prepared from New Zealand white rabbit skeletal muscle according to a previously published method [21]. Skeletal ryanodine receptor (RyR1) was purified as previously described [3].

    • Tilapia and human CLIC2 structures are highly conserved

      2018, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Since Xq28 is the region associated with several human diseases such as mental retardation and X-linked epilepsy, human CLIC2 has thus been proposed to be one of the candidate genes related to these diseases [7,8]. So far, the known interaction partners of human CLIC2 are cardiac ryanodine receptors 1 and 2 (RyR1 and RyR2), whose activities could be reversibly inhibited by CLIC2 [9–11]. The ability of soluble CLIC2 to form chloride channels has been confirmed by in vitro experiments, suggesting that CLIC2 belongs to one of the Janus proteins [6].

    • Physiology and Pharmacology of Ryanodine Receptor Calcium Release Channels

      2017, Advances in Pharmacology
      Citation Excerpt :

      The effect of CLIC2 on RyR activity is complex as it inhibits RyR2 when the redox potential is oxidizing, but activates channels under more reducing conditions (Jalilian, Gallant, Board, & Dulhunty, 2008). CLIC2 potentiates [3H]ryanodine binding, but inhibits RyR channels and Ca2+ release from skeletal and cardiac SR, questioning the dogma that [3H]ryanodine-binding simply reflects RyR open probability (Dulhunty, Pouliquin, Coggan, Gage, & Board, 2005; Meng et al., 2009). Finally, CLIC2 enhances substate activity in individual RyRs and increases coupled gating events, where several channels in the bilayer open simultaneously (Dulhunty, Pouliquin, et al., 2005).

    View all citing articles on Scopus

    X.M., G.W. and C.V. contributed equally to this work.

    View full text