Muscle physiology and biochemistry

MUSCLE PHYSIOLOGYAND BIOCHEMISTRY Muscle Physiology and Biochemistry Edited by SHOICHIIMAI MAKOTOENDO Department of Pharmacology Niigata University School of Medicine No 757, Asahimachi-dori Niigata 951, Japan Saitama Medical School Moroyamamachi Saitama 350-04 Japan IWAO OHTSUKI Faculty ofMedicine Kyushu University Fukuoka 812-82 Japan Reprinted from Molecular and Cellular Biochemistry, Volume 190 (1999) Springer-Science+Business Media, B.V Library of Congress Cataloging-in-Publication Data Musc1e physiology and biochemistry/edited by Shoichi Imai, Makoto Endo, Iwao Ohtsuki p cm (Developments in molecular and cellular biochemistry) ISBN 978-1-4613-7534-0 ISBN 978-1-4615-5543-8 (eBook) DOI 10.1007/978-1-4615-5543-8 Musc1es Physiology Musc1e contract ion Musc1es- -Molecular aspects Musc1es Metabolism Imai, Shoichi, 1931- II Endo, Makato 1933- III Ohtsuki, Iwao IV Series QP321.M8917 1998 616.7' dc21 98-36698 ISBN 978-1-4613-7534-0 Printed an acid-free paper Ali rights reserved © 1999 Springer Science+Business Media Dordrecht Originally published by K1uwer Academic Publishers in 1999 Softcover reprint of the hardcover 1st edition 1999 No part ofthe material protected by this copyright notice may be reproduced or utilized in any farm or by any means, electronic or mechanical, inc1uding photocopying, recarding ar by any information storage and retrieval system, without written permis sion from the copyright owner Molecular and Cellular BiochelDistry: An International Journal for Chemical Biology in Health and Disease CONTENTS VOLUME 190, Nos & 2, January (I) 1999 MUSCLE PHYSIOLOGY AND BIOCHEMISTRY Shoichi Imai, Makoto Endo and Iwao Ohtsuki Preface M Endo: Dedication J Gergely: Professor Ebashi' s impact on the study ofthe regulation of striated muscle contraction S.Y Perry: Troponin I: Inhibitor or facilitator I Ohtsuki: Calcium ion regulation ofmuscle contraction: The regulatory role oftroponin T K Yamada: Thermodynamic analyses ofcalcium binding to troponin C, calmodulin and parvalbumins by using microcalorimetry M Yazawa, K.-i Nakashima and K Yagi: A strange calmodulin of yeast A.G Szent-Gyorgyi, Y.N Kalabokis and C.L Perreault-Micale: Regulation by molluscan myosins Y Yazawa and M Kamidochi: The properties and function of invertebrate new muscle protein A Weber: Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms M Tanokura and Y Suzuki: A phosphorus-31 nuclear magnetic resonance study on the complex ofchicken gizzard myosin subfragment I with adenosine diphosphate DJ Hartshorne and K Hirano: Interactions of protein phosphatase type 1, with a focus on myosin phosphatase K Fujita, L.-HYe, M Sato, T Okagaki, Y Nagamachi and K Kohama: Myosin light chain kinase from skeletal muscle regulates anATP-dependent interaction between actin and myosin by binding to actin T Murahashi,A Fujita and T Kitazawa: Ca 2+-induced Ca 2+ desensitization ofmyosin light chain phosphorylation and contraction in phasic smooth muscle T Masuda, K Ohmi, H Yamaguchi, K Hasegawa, T Sugiyama, Y Matsuda, M lino and Y Nonomura: Growing and differentiating characterization ofaortic smooth muscle cell line, p53LMACO obtained from p53 knock out mice K Sobue, K Hayashi and W Nishida: Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation I Niki and H Hidaka: Roles of intracellular Ca2+ receptors in the pancreatic ~-cell in insulin secretion Y Soeno, H Yajima, Y Kawamura, S Kimura, K Maruyama and T Obinata: Organization ofconnectinltitin filaments in sarcomeres of differentiating chicken skeletal muscle cells K.-i Kusano, H Abe and T Obinata: Detection of a sequence involved in actin-binding and phosphoinositide-binding in the Nterminal side ofcofilin E Ozawa, Y Hagiwara and M Yoshida: Creatine kinase, cell membrane and Duchenne muscular dystrophy T Masaki, H Ninomiya,A Sakamoto and Y Okamoto: Structural basis of the function of endothelin receptor Y Yoshida,A Toyosato, M.O Islam, T Koga, S Fujita and S Imai: Stimulation of plasma membrane Ca 2+-pump ATPase ofvascular smooth muscle by cGMP-dependent protein kinase: Functional reconstitution with purified proteins H Yamamoto and M Kawakita: Chemical modification ofan arginine residue in theATP-binding site ofCa2+-transportingATPase of sarcoplasmic reticulum by phenylglyoxal M Hirata, M Yoshida, T Kanematsu and H Takeuchi: Intrinsic inhibitor of inositol I ,4,5-trisphosphate binding M lino: Dynamic regulation of intracellular calcium signals through calcium release channels Y Ogawa, T Murayama and N Kurebayashi: Comparison of properties ofCaH release channels between rabbit and frog skeletal muscles Index to Volume 190 I 3-4 5-S 9-32 33-38 39-45 47-54 55-62 63-66 67-74 75-78 79-84 85-90 91-98 99-104 105-118 119-124 125-131 133-141 143-151 153-156 157-167 169-177 179-184 185-190 191-201 203-204 Molecular and Cellular Biochemistry 190: 1, 1999 Preface The papers in this issue were contributed by close friends, coworkers and pupils of Professor Setsuro Ebashi They are dedicated to him to commemorate his great and pioneering contribution to the advancement of muscle physiology and biochemistry, which in course of time exerted a great influence on the whole field of life science We would like to express our cordial thanks to an the contributors who made the publication of this issue possible Owing to some unexpected troubles of one ofthe editors (M E.) the publication of this issue has been greatly delayed, for which he sincerely apologizes to all the contributors and other editors We believe that this issue reveals the present state of research on muscle and/or calcium that had been opened up by Professor Ebashi Shoichi Imai, Niigata, Japan Makoto Endo, Saitama, Japan Iwao Ohtsuki, Fakuoka, Japan Molecular and Cellular Biochemistry 190: 3-4, 1999 Dedication Setsuro Ebashi was born in Tokyo on 31 st August, 1922 There is a Japanese saying that 'Sandalwood is fragrant even in seed leaf.' Genius displays itself even in childhood Finishing the six-year course ofprimary school in five years and the five-year course of middle school in four years, he entered the First High School, the most prestigious high school in Japan, at the age of only 15 In July 1942, when he was an undergraduate student of Faculty of Medicine, Tokyo Imperial University (now called University ofTokyo), he by chance visited the laboratory of Dr Hiroshi Kumagai, at that time Lecturer in Pharmacology, to have a practical training during the summer vacation This was the beginning of an admirable relationship of love and kindness between a pupil and a teacher as well as the start of Dr Ebashi' s muscle research However, the War severed the relationship: the teacher went to Indonesia to teach in Jakarta Medical School, and the pupil received his M.D degree in 1944 and served in the war as a naval surgeon When Dr Ebashi was demobilized in 1946, he went straight to Dr Kumagai's laboratory again Dr Ebashi's research was at first electrophysiology of smooth muscle in which Dr Kumagai had a deep interest However, in 1950 Dr Ebashi was deeply impressed with a J Physiol paper by Hodgkin and Katz (1949) which completely elucidated the mechanism of excitation as he felt At about the same time he was also deeply inspired by a book 'Chemistry of Muscular Contraction' by A Szent-Gyorgyi (1949) These readings led him to change the subject of his research to the contractile mechanisms He raised the following question Although Szent-Gyorgyi demonstrated thatATP added to the actin-myosin system such as actomyosin thread or glycerinated muscle induces contraction, removal ofATP does not cause relaxation, which is quite different from, for example, acetylcholine-induced contraction of living muscle, where the removal of acetylcholine causes relaxation His idea was that there must be something in living muscle to cause relaxation, which was lost and absent in the actomyosin systems He started to search for the relaxing factor in homogenized muscle and soon he found the factor and reported to a meeting of a Japanese muscle physiology group in 1952 Sometime after this Dr Kumagai found a paper by Marsh in Nature (1951) that had already reported the same factor However, this was not a disappointment for young Dr Ebashi but rather an encouragement because it proved that his direction of research was right Having inquired further into the relaxing factor, he demonstrated in 1955 that the essential component of the relaxing factor was in the particulate fraction, against the general beliefat that time that it may beATP-regenerating soluble enzyme(s) As for the mechanism of relaxation by the relaxing factor, once again against the general belief at that time that the relaxing factor might produce some (organic) substance which in tum acts on the actomyosin system to cause relaxation, Dr Ebashi showed in the early 60s that removal ofCa 2+ ion from the medium by the relaxing factor is the cause of relaxation His evidence consisted oftwo important discoveries that the particulate relaxing factor strongly accumulates Ca2+ ion from the medium in the presence ofATP, and that a minute amount of Ca 2+ ion is necessary for the contractile reaction of well-washed Ca2+-free natural actomyosin system Although physiologists had recognized the contractioninducing action of Ca2+ ion, it had not been recognized by muscle biochemists before Dr Ebashi, because all the biochemical experiments were done in the presence of sufficient amount of Ca2+ion contaminated from reagents or exuded from glasswares Dr Ebashi further demonstrated electronmicroscopically that the relaxing factor has a vesicular structure, indicating that it is the fragment ofthe sarcoplasmic reticulum (SR) Since relaxation is the reverse of Molecular and Cellular Biochemistry 190: 5-8, 1999 © 1999 Kluwer Academic Publishers Professor Ebashi's impact on the study of the regulation of striated muscle contraction John Gergely Muscle Research Group, Boston Biomedical Research Institute; Department ofNeurology, Massachusetts General Hospital; Department ofBiological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Abstract The field of striated muscle regulation has changed tremendously over the last forty years Many of the problems solved by Dr Ebashi and by those stimulated by him offer new challenges for future generations of scientists Many questions remain to be solved, and it should give particular pleasure to Dr Ebashi to see how the seeds sown by him and his col1eagues have now grown into a beautiful tree that bears rich fruit at present and will continue to so for a long time in the future (Mol Cel1 Biochem 190: 5-8, 1999) Key words: troponin, tropomyosin, thin filament regulation, Ca 2+ Introduction I am indeed greatly pleased and honored to be able to join in the celebration of the remarkably productive and influential contribution of Professor Ebashi's life in science This gives me particular pleasure since so much of what my colleagues and I were able to in the last four decades has depended on his contributions Dr Ebashi became known very early in his career by establishing the nature ofthe so-called relaxing factor This was followed by the identification of calcium ions as key messengers in the activation process of muscle contraction and the discovery of the troponin complex which, together with tropomyosin, was identified as the actin-bound regulatory system of striated muscle (See [I]) In this brief review (For detailed reviews see: [1,52-57]) I should like to trace some of the developments concerning the regulation of striated muscle that were sparked by the ground-breaking work of Dr Ebashi and his colleagues and to point to some questions that currently await answers The discovery of troponin The new component discovered in Dr Ebashi's laboratory was first known as native tropomyosin [2,3] It rendered the interaction of actin and myosin in the presence of ATP Ca2+ sensitive Native tropomyosin was soon separated into two components: tropomyosin and a new entity that became known as troponin Troponin was identified as the receptor for Ca 2+, whose role in actomyosin activation had earlier been established [4, 5] with four Ca-binding sites in troponin [6] There were indications that troponin is a multi-component system [7] The work initiated in the Ebashi laboratory started a new era in muscle research involving many laboratories all over the world It became clear that there was a calcium binding component and another one that inhibited the Mg 2+ stimulatedATPase ofpurified actomyosin [8,9] By 1972 a general consensus was reached that troponin consists of three subunits [10-13] whose names indicate their roles: viz troponin C (TnC), troponin I (Tnl), and troponin T (TnT) for calcium binding, inhibition, and troponin binding, respectively The availability of purified components oftroponin made it possible to build on the findings that emerged in the earlier stages Calcium binding studies [14], utilizing the calcium buffer system originally developed by Dr Ebashi and his colleagues [5], located four calcium binding sites in TnC and showed that there are two classes each containing two calcium binding sites Two sites bind calcium with high affinity, as well as Mg2+ although with lower affinity, while the other two sites ofTnC are essentially specific for calcium Address/or offprints: J Gergely, Muscle Research Group, Boston Biomedical Research Institute, 220 Staniford Street, Boston, MA 02114-2500, USA Troponin C - The Ca2+ receptor While research on troponin began to flourish studies on a related protein led to important results Parvalbumin, which had first been found in fish muscle as a cytoplasmic rather than a myofibrillar protein, was characterized both in terms of primary structure and crystal structure as having two Ca 2+ binding sites [15] This work led to the concept of the so-called EF hands suggested by a bent middle finger, the thumb and index finger as depicting a calcium binding loop flanked by two a helices as the model of calcium binding sites originally found in parvalbumin and by now known to occur in large super families of Ca 2+binding proteins When the amino acid sequence ofTnC became known [16] a high degree of homology with parvalbumin was recognized and the Ca2+binding sites were identified A variety ofstudies have shown that sites I and II in the N-terminal domain are Ca-specific sites; sites III and IV are the high affinity Ca-Mg sites in the C domain The former are recognized as the functionally important triggering sites (see [17]) and references therein) The similarities between the TnC and parvalbumin structures led to speculations about how two parvalbumin-like halves could be fitted into the structure ofTnC [18] When the structure ofTnC was solved by x-ray crystallography [19, 20], it showed two domains - each a parvalbumin-like structure connected, however, by a single a helix instead of a compact molecule essentially containing two paravalbumin-like structures The molecular switch in troponin C An important step toward our current understanding of the chain of events initiated by calcium binding to the triggering sites in TnC came from insights ofHerzberg et al [21] gained in comparing the structure of the N- and C-terminal homologous domains in TnC Owing to the conditions of crystallization the former contained no bound calcium, while the latter had two sites occupied by Ca 2+ Thus the difference between the two domains would give a clue to the conformational changes brought about by calcium when it becomes bound to the N terminal sites This led to the suggestion that the connector between helices Band C together with the link between them moves away from helix D which is part ofthe long helix connecting the two domains, exposing a hydrophobic area which was presumed to become an interacting site with Tn! Soon thereafter various pieces of evidence emerged for this view Site directed mutagenesis of charged residues [22] or disulfide formation between genetically engineered Cys residues [23], in segments whose separation was expected to change upon Ca 2+ binding according to the model, led to changes in Ca 2+-binding and ATPase activity Distance determinations by resonance energy transfer between probes on appropriately placed engineered Cys residues showed a Ca 2+-induced change corresponding to the expectations based on the model [24] Finally, solution ofthe high resolution NMR structure ofTnC with four Ca2+ bound [25] brought definitive proof for the postulated structure The opening of the N terminal domain of TnC may be considered as the molecular switch in TnC The NMR structure revealed some differences between helix B in the N-terminal domain and the corresponding helix in the C terminal domain even when both sites in each domain were occupied by Ca2+ It also pointed to some flexibility of the central helix in solution A recent comparison ofthe high resolution NMR structures of cardiac and skeletal TnC in the 4-Ca2+ state shows that the extent of opening of the hydrophobic surface is much less in the case of cardiac muscle, a finding whose full implications are yet to be explored [26] The opening of the N terminal domain of TnC may be considered as the molecular switch in TnC The molecular switch in troponin I The next question that has received some partial answers over the years is the status of the molecular switch in Tn! In the absence ofhigh resolution structures for TnI and its complexes such answers must remain tentative There is evidence that portions of Tnl move under the influence of activation from TnC to actin, and under conditions corresponding to relaxation they return to TnC One of the sites that has been used is cysteine 133 [27] and current studies are further exploring movements in Tnl by labeling cysteine residues introduced by genetic engineering as has been done in the case ofTnC There are some not fully answered questions concerning the relation of the region that comes into close contact with actin and the so-called inhibitory region that emerged in earlier studies and contains the stretch of residues 96-116 in Tnl [28] Evidence is accumulating that this inhibitory region is indeed interacting with both domains of TnC [29-31] but its mode of interaction with actin needs further elucidation During recent years a reasonable consensus has emerged concerning the overall arrangement of the polypeptides in TnC- and Tnl relative to each other Both crosslinking [32, 33] and fragment binding [34] studies suggest that the two chains run in opposite directions; that is, the N terminus of TnC interacts mainly with the C terminus of TnI and vice versa However, evidence is also at hand indicating that within Tnl certain stretches may run locally in opposite directions while the overall trend is preserved Recent work on troponin with Tnl containing only Cys 133 and Cys 48 thiols for placement of probes for resonance energy transfer studies has shown metal dependent conformational changes in Tnl modulated by the interaction oftroponin with actin [35] Research on certain aspects of TnI/TnC interaction has been stimulated by studies on calmodulin, which is an activator of a large number of enzymes In light of the close similarities between TnI and calmodulin with respect to their chemical and crystallographic structure the question arises whether the structural changes occurring when TnI binds to TnC are similar to those taking place on the interaction of calmodulin with one of its target proteins In the case of calmodulin, both x-ray diffraction [36] and multidimensional NMR [37] studies showed that at least with the M 13 peptide derived from myosin light chain kinase - a well known target of calmodulin playing a role in the activation of smooth muscle contraction - is accompanied by a large structural change in calmodulin bringing the two globular domains homologous to those in TnC close together As far as the TnI'TnC complex is concerned, evidence points in the opposite direction indicating an essentially extended structure for TnC based both on resonance energy transfer distance determinations [38] and low angle x-ray and neutron diffraction studies [39,40] The latter studies also point to the existence of masses derived from TnI beyond the Nand C terminal domains of TnC, a picture whose details remain to be filled in in terms of the course of the polypeptide chain in Tn! Students of this field are eagerly awaiting a more definitive x-ray crystallographic study of the intact troponin complex Very recently crystallization of a complex between an N-terminal fragment of TnI and TnC has been achieved and the high resolution structure derived from x-ray diffraction reported [41] This work provides some interesting interim results pending the availability of crystals of the full complex Troponin T Although the shape of TnT has been long established starting with the immuno-electron microscopic demonstration that the globular C-terminal portion and the highly a-helical Nterminal portion of TnT occupy distinguishable sites along tropomyosin [42], little is known about the role of TnT in the mechanism of regulation While early work has considered TnT mainly as an anchor tying the rest of the complex to tropomyosin, hence the suffix T to troponin, recent evidence assigns a more active role to TnT serving as a signal transmitter between TnC and TnI [43] as well as modulating the effect of myosin heads on the activity ofthe troponin complex [44) Filament regulation and cooperativity From the earliest days of the identification of the participants in the regulatory machinery, viz troponin and tropomyosin, the question of how changes in solution are related to those in the actin filament itself have been intriguing (see [45] and references therein) Recent x-ray diffraction studies on reconstituted actomyosin gels and on muscle fibers have thrown new light on tropomyosin movement associated with Ca 2+ activation [46-48) It appears that in the regulated thin filament Ca 2+ binding to troponin is accompanied by a movement about 30° azimuthally towards the central groove from a position where it would block strong myosin binding, according to the current model of myosin-actin interactions (see [49]) Binding of myosin, which would take place to a site partially unblocked by calcium, causes a small but significant further change in the position of tropomyosin, consistent with a cooperative role of myosin - first pointed to by A Weber and her colleagues [50] - in the full activation of the thin filament This two step model of activation seems to be in harmony with the three state model based on kinetic studies in solution, the third state being the Ca 2+ free, 'blocked' state [51] References I Ebashi S, Endo M: Calcium ion and muscle contraction [Review] Prog Biophys Mol Bioi 18: 123-183,1968 Ebashi S: Third component participating in the precipitation of 'natural actomyosin' Nature 200: 1010, 1963 Ebashi S, Ebashi F: A new protein component participating in the superprecipitation of myosin J Biochem 55: 604-613,1964 Weber A: On the role of calcium in the activity of adenosine-5'triphosphate hydrolysis by actomyosin J Bioi Chern 234: 2764-2769, 1959 Ebashi S: Calcium binding and relaxation in the actomyosin system J Biochem 48: 150 -151, 1960 Ebashi S, Ebashi F, KodamaA: Troponin as the Ca 2+-receptive protein in the contractile system J Biochem 62: 137-138, 1967 Ebashi S, Wakabayashi T, Ebashi E: Troponin and its components J Biochem 69: 441-445, 1971 Hartshorne OL, Perry SV, Schaub MC: A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized actomyosin Biochem J 104: 907-913,1967 Hartshorne OJ, Mueller H: Fractionation oftroponin into two distinct proteins Biochem Biophys Res Comm 31: 647 653, 1968 10 Greaser ML, Gergely J: Reconstitution oftroponin activity from three protein components J Bioi Chern 246: 4226-4233, 1971 II Greaser ML, Gergely J: Purification and properties of the components from troponin J Bioi Chern 248: 2125-2133,1973 12 Ebashi S: Separation oftroponin into its three components J Biochem 72: 787-90, 1972 13 Perry SV, Cole HA, Head JF, Wilson FL: Localization and mode of action of the inhibitory protein component of the troponin complex Cold Spring Harbor Symp Quant Bioi 37: 251-262, 1972 14 Potter JO, Gergely J: The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase J BioI Chern 250: 4628-4633, 1975 15 Kretsinger RH, Nockolds CE: Carp muscle calcium binding protein II Structure determination and general description J Bioi Chern 248: 3313-3326, 1973 16 Collins lH, Potter lD, Hom Ml, Wilshire G, lackman N: The amino acid sequence of rabbit skeletal muscle troponin C: Gene replication and homology with calcium-binding proteins from carp and hake muscle FEBS Lett 36: 268-272, 1973 17 Grabarek Z, Drabikowski W, Leavis PC, Rosenfeld SS, Gergely L: Proteolytic fragments oftroponin C Interactions with the other troponin subunits and biological activity Bioi Chern 256: 13121-13127, 1981 18 Kretsinger RH, Barry CD: The predicted structure ofthe calcium-binding component oftroponin Biochim Biophys Acta 405: 40-52,1975 19 Sundaralingam M, Bergstrom R, Strasburg G, Rao ST, Roychowdhury P, el al.: Molecular structure oftroponin C from chicken skeletal muscle at 3-angstrom resolution Science 227: 945-948, 1985 20 Herzberg 0, lames MN: Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution Nature 313: 653-659, 1985 21 Herzberg 0, Moult 1, lames MN: A model for the Ca'+-induced conformational transition of troponin C A trigger for muscle contraction Bioi Chern 261: 2638-2644, 1986 22 Fujimori K, Sorenson M, Herzberg 0, Moult 1, Reinach FC: Probing the calcium-induced conformational transition of troponin C with site-directed mutants Nature 345: 182-184, 1990 23 Grabarek Z, Tan RY, Wang 1, Tao T, Gergely J: Inhibition of mutant troponin C activity by an intra-domain disulphide bond Nature 345: 132-135,1990 24 Wang Z, Gergely 1, Tao T: Characterization of the Ca'+-triggered conformational transition in troponin C Proc Natl Acad Sci USA 89: 11814-11817, 1992 25 Siupsky CM, Sykes BD: NMR solution structure of calcium-saturated skeletal muscle troponin C Biochemistry 34: 15953-15964, 1995 26 Gagne SM, Li MX, McKay RT, Sia SK, Spyracopoulos L, el al.: The calcium induced structural change in that triggers skeletal and cardiac muscle contraction Biophys 72: A332, 1997 27 Tao T, Gong BJ, Leavis PC: Calcium-induced movement oftroponin-l relative to actin in skeletal muscle thin filaments Science 247: 13391341,1990 28 Syska H, Wilkinson 1M, Grand RJ, Perry SV: The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit Biochem J 153: 375-387, 1976 29 Leszyk 1, Grabarek Z, Gergely J, Collins JH: Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: Evidence for direct interaction between the inhibitory region oftroponin I and the NH,- terminal, regulatory domain oftroponin C Biochemistry 29: 299-304, 1990 30 Pearlstone lR, Smillie LB: Evidence for two-site binding oftroponin I inhibitory peptides to the Nand C domains of troponin C Biochemistry 34: 6932-6940, 1995 31 Pearlstone JR, Sykes BD, Smillie LB: Interactions of structural Cdomain and regulatory N-domains of troponon C with repeated sequence motifs in troponin I Biophys J 72: A331, 1997 32 Kobayashi T, Tao T, Gergely J, Collins 1: Structure of the troponin complex Implications of photocross-linking oftroponin I to troponin C thiol mutants Bioi Chern 269: 5725-5729, 1994 33 lha PK, Mao C, Sarkar S: Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: The inhibitory region enhances binding oftroponin I fragments to troponin C Biochemistry 35: 11026-11035, 1996 34 Farah CS, Miyamoto CA, Ramos C, Dasilva A, Quaggio RB, el al.: Structural and regulatory functions of the NH,- and COOH-terminal regions of skeletal muscle troponin I J Bioi Chern 269: 5230-5240, 1994 35 Luo Y, Wu l-L, Gergely 1, Tao T: Troponin T and Ca2+ dependence of the distance between Cys48 and Cys 133 of troponin I in the ternary troponin complex and reconstituted thin filaments Biochemistry 36: 11027-11035,1997 36 Meador WE, Means AR, Quiocho FA: Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex Science 257: 1251-1255,1992 37 Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A: Solution structure of a calmodulin-target peptide complex by multidimensional NMR Science 256: 632-638, 1992 38 Gong B-1, Wang Z, Tao T, Gergely J: Troponin C remains extended in the ternary trroponin complex Biophys 66: A346, 1994 39 Olah GA, Rokop SE, Wang C-LA, Blechner SL, Trewhella J: Troponin I encompasses an extended troponin C in the Ca'+-bound complex - a small-angle X-ray and neutron scattering study Biochemistry 33: 8233-8239, 1994 40 Olah GA, Trewhella J: A model structure ofthe muscle protein complex 4-Ca'+-troponin C-troponin I derived from small-angle scattering data - implications for regulation Biochemistry 33: 12800-12806, 1994 41 Vassylyev DG, Takeda S, Wakatsuki S, Maeda K, Maeda, Y: Crystal structure of troponin C in complex with troponin I fragment at 2.3A resolution Proc Natl Acad Sci (USA) 95: 4747 4852, 1998 42 Ohtsuki I: Molecular arrangement oftroponin-T in the thin filament J Biochem 86: 491 497,1979 43 Potter JD, Sheng Z, Pan BS, Zhao J: A direct regulatory role for troponin T and a dual role for troponin C in the Ca'+ regulation of muscle contraction BioI Chern 270: 2557-2562, 1995 44 Schaertl S, Lehrer SS, Geeves MA: Separation and characterization of the two functional regions of troponin involved in muscle thin filament regulation Biochemistry 34: 15890-15894, 1995 45 Potter JD, Gergely 1: Troponin tropomyosin and actin interactions in the Ca2+ regulation of muscle contraction Biochemistry 13:2697-2703, 1974 46 Lorenz M, Poole KJV, Popp D, Rosenbaum G, Holmes KC:An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels J Mol Bioi 246: 108119,1995 47 Poole KJV, Evans G, Rosenbaum G, Lorenz M, Holmes KC: Thc effect of crossbridges on the calcium sensitivity of the structural change in the regulated thin filament Biophys J 68: A365, 1995 48 Holmes KC: The actomyosin interaction and its control by tropomyosin Biophys 168: S2-S7, 1995 49 Rayment 1, Holden HM, Whittaker M, Yohn CB, Lorenz M et al.: Structure of the actin-myosin complex and its implications for muscle contraction Science 261: 58-65, 1993 50 Bremel RD, Murray JM, Weber A: Manifestations of cooperative behavior in the reglated actin filament during actin activated ATP hydrolysis in the presence of calcium Cold Spring Harbor Symp Quant BioI 37: 267-275,1972 51 McKillop D, Geeves MA: Regulation of the interaction between actin and myosin subfragment-I - evidence for states of the thin filament Biophys J 65: 693-701,1993 52 Leavis PC, Gergely 1: Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction CRC Crit Rev Biochem 16: 235-305,1984 53 Ohtsuki 1, Maruyama K, Ebashi S: Regulatory and cytoskeletal proteins of vertebrate skeletal muscle Adv Prot Chern 38: 1-67, 1986 54 Zot AS, Potter lD: Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction Ann Rev Biophys Biophys Chern 16: 535-559, 1987 55 Chalovich 1M: Actin mediated regulataion of muscle contraction Pharmac Ther 55: 95-148,1992 56 Farah CS, Reinach FC: The troponin complex and regulation of muscle contraction [Review] FASEB 19: 755-767, 1995 57 Tobacman LS: Thin Filament-mediated Regulation of Cardiac Contraction [Review] Ann Rev Physiol58: 447 481,1996 Molecular and Cellular Biochemistry 190: 191-201, 1999 © 1999 Kluwer Academic Publishers Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles Yasuo Ogawa, Takashi Murayama and Nagomi Kurebayashi Department ofPharmacology, Juntendo University School ofMedicine, Tokyo, Japan Abstract Biochemical investigation of Ca2+release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation ofCa2+release In this review, we compared the properties ofryanodine receptors (RyR), Ca2+release channel protein, in skeletal muscles between rabbit and frog While the Ryrl isoform is the main RyR of rabbit skeletal muscles, two isoforms, (X- and ~-RyR which are homologous to Ryrl and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum CICR and pH]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca 2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences First, rabbit RyR showed higher Ca2+affinity than the frog Second, rabbit RyR showed higher activity in the presence of Ca 2+ alone with less stimulation by adenine nucleotide than the frog Third, rabbit RyR displayed less enhancement of pH]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+concentrations in the M NaCI medium, while frog RyR showed disinhibition Other matters relevant to Ca2+release were also discussed (Mol Cell Biochem 190: 191-201,1999) Key words: Ca2+release, frog, rabbit, ryanodine receptor, skeletal muscle Introduction Professor Ebashi always stressed to his students that biochemical investigation ofmuscle proteins was carried out with rabbit skeletal muscles, while frog skeletal muscle had been preferred for physiological approaches to muscle contraction, and that quantitative investigation of calcium ion balance was necessary to establish the regulatory role of calcium ion in muscle contraction One of the authors (YO), when a postgraduate student, had a chance to help Dr S Winegrad who was attempting to prepare an entire sarcoplasmic reticulum (SR) from frog skeletal muscle in Dr Ebashi's laboratory during the latter's sabbatical leave Frog skeletal muscle was, thereafter, the primary material used in our experiments There are few laboratories except ours where biochemical investigation of SR from frog skeletal muscles has ever been routinely conducted Many investigations of isolated Ca2+- release channel or protein, i.e ryanodine receptor RyR, have been made with rabbit skeletal muscle, and it is generally assumed that the property ofRyR from rabbit might hold with other species We noticed that this was not the case in some respects with frog RyR We discuss in this article the similarity and the difference ofCa 2+release channels between rabbit and frog skeletal muscle General aspects ofRyR can be found in the many reviews already published [1-9] Some differences in Ca 2+-uptake by SR between rabbit and frog have been discussed elsewhere [10] The principal component of Ca 2+-release channels The Ca2+-release channel in skeletal muscle is largely composed oftetramer ofryanodine receptor (RyR), a protein of Address for offprints: Y Ogawa, Department ofPharmaco!ogy, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113, Japan 192 about 5000 amino acid residues with a molecular weight of about 560 kDa Three different genes express the corresponding isoforms in mammals: Ryr by ryr J, Ryr2 by ryr2 and Ryr3 by ryrJ [6,9] The protein-staining pattern of SR on SDS-PAGE showed a single band of RyR with rabbit skeletal muscle in contrast to double bands with frog skeletal muscle [11, 12] The RyR in rabbit skeletal muscles turned out to be Ryrl Double bands of similar density with frog skeletal muscle are referred to as a- and /3-RyR isoforms The two isoforms occur as a homotetramer in a muscle fiber [12, 13]; their coexistence occurs not only in frog skeletal muscles but also in chicken, and fishes [11, 14, 15] They are distinct proteins, and /3-RyR, which shows the greater mobility, is not a degradation product of a-RyR [12,13] /3-RyR is not a homologue of Ryr2, although this was originally the prevailing expectation among many investigators [16, 17]: Sutko and his colleagues [18] showed a third distinct isoform in avian cardiac muscle, and no positive band was observed on Western blot analysis with anti-/3-RyR antibody of microsomes from bullfrog cardiac muscle [14] Sequences of the cloned cONAs showed that a- and /3-RyR of frog skeletal muscle are most homologous to mammalian Ryrl and Ryr3, respectively [19] Recently a full amino acid sequence for /3-RyR of chicken skeletal muscle was deduced, while that for a-RyR remains only partial [20] We can conclude, however, that a- and /3-RyR of chicken skeletal muscle are most homologous to mammalian Ryrl and 3, respectively, as true offrog skeletal muscle We can further assume that most skeletal muscles from non-mammalian vertebrates have two isoforms (a- and /3-RyRs, i.e., Ryrl and homologues) in similar amounts in the same fiber Not all skeletal muscles from non-mammalian vertebrates have two coexisting isoforms: lizards and snakes showed a single band ofa-RyR, while turtles and crocodiles showed a- and /3-RyRs as did fishes, amphibians and birds [15] Extraocular and swimbladder muscles from fishes which contract very rapidly showed only a-RyR while their body skeletal muscle had both a- and /3-RyR The rattlesnake tail-shaker muscle which produces sound by rapid vibration as the swimbladder muscles also has a single isoform, a-RyR [15] Caffeine sensitive Ca2+-release was detected in skeletal muscle cells from ryr I-gene targeted mouse, and this was ascribed to Ryr3 because mRNA from ryrJ was recognized [21,22] The detection of Ryr3 in normal skeletal muscles by Western blot analysis, however, has been unsuccessful to date except by Sorrentino's laboratory He estimated that Ryr3 accounted for one twentieth to one-fiftieth of Ryr1 in mammalian skeletal muscle [23, 24] The contents of Ryr3 varied among different muscles, e.g relatively higher in diaphragm and soleus, and lower levels in abdominal muscles and tibialis anterior No detectable levels of Ryr3 were observed in the extensor digitorum longus [24] Murayama and Ogawa [25] estimated by immunoprecipitation that Ryr3 in the rabbit brain, a well-known expression site of Ryr3, was less than 2% of total RyR (mostly Ryr2) in that organ They recently obtained results with rabbit diaphragm that the fraction of Ryr3 content in the muscle was much less than its value in the brain [26] In the back muscles no Ryr3 was detected by immunoprecipitation This may not be consistent with the hypothesis that rapid contraction requires the presence of Ryrl alone Rigorous re-examination will be necessary to determine whether nonmammalian skeletal muscles that showed a single band ofa-RyR on SOS-PAGE have, in fact, no /3-RyR or too little to detect it by conventional analysis We have mentioned above that a- and /3-RyRs form homotetramers and function as Ca 2+-induced Ca2+ release (CICR) channels Ryr3 also functions as a homotetramer not only in rabbit brain [25] but also in skeletal muscles [26] Preliminary results in our work showed that Ryr3 coexisted with Ryrl in the same cells of skeletal muscle fibers Formation of a homotetramer even in as small a fraction as below 2%, but with no evidence of a heterotetramer, is in marked contrast to the inositol trisphosphate receptor, another Ca2+ release channel protein The potential formation of a heterotetramer of inositol trisphosphate receptor (at least isoforms) may be an underlying mechanism accounting for its diverse properties [27,28] The content of Ryr3 may be far less than 2% of Ryr I, at least in rabbit skeletal muscle The difference in [3H]ryanodine binding activity between Ryrl and Ryr3 was not marked enough to compensate for the difference in their contents [25, 26] Putative Ryr3 from ryr I-targeted mouse was, in fact, reported to be about 20 times lower in Ca 2+sensitivity in Ca 2+induced Ca 2+release (CICR) [22] Therefore, the contribution of Ryr3 to CICR from and FH]ryanodine binding to SR must be negligible in rabbit skeletal muscle The involvement of Ryr3 in excitation-contraction coupling is also probably minor, if any This conclusion is consistent with the finding that the skeletal muscles from ryrJ-knocked out mouse showed contractions similar to those from a normal animal, while those from ryr I-targeted mouse did not contract on depolarization [21,29] Correspondingly, a-RyR, the homologue to Ryrl, is likely to play an important role in the excitation-contraction coupling in nonmammalian vertebrates, because skeletal muscles from a crooked-neck dwarf mutant chick which lacks a-RyR failed to contract on depolarization [30] Because those mutant muscles showed Ca 2+release on application of caffeine, /3-RyR is functional as a CICR channel In view of the characteristic disposition of Ryrl to which putative voltage sensors are alternately apposed in swimbladder muscles [31] and mammalian skeletal muscles [4], the disposition of a- and /3-RyR, i.e., alternate or random arrays in nonmammalian vertebrates is of interest The biological significance of /3-RyR which is the homologue to Ryr3 and occurs in a similar amount remains to be elucidated 193 Ca2+-induced Ca2+ release (CICR) and RyR General properties of CICR Since the first demonstration ofCICR by Ford and Podolsky [32] and Endo et al [33], CICR has been energetically studied; it is a Ca2+ release mechanism not only in skeletal muscles but also in various kinds of cells including other types of muscle cells However, there are two different opinions about the biological role of CICR in the physiological contraction ofskeletal muscle: one is that CICR serves as an amplifier of Ca 2+ release triggered by conformation change of the voltage sensor (i.e., the dihydropyridinederivative receptor), and the other is that CICR has no function in the physiological depolarization-induced contraction [34-37] Because an understanding of CICR is helpful in clarifying the properties of RyR, we offer a summary of the findings on CICR to date Figure shows CICR in skinned frog iliofibularis muscle fibers The rates of CICR triggered by Ca2+ alone were very small (triangles) However, the addition of an adenine nucleotide, mM AMP in this case (circles), enhanced these rates as much as 100 times without significant change in their Ca + dependency Endo [38] reported that the factor of enhancement could reach as high as 1000 The rate increased with increase in the Ca2+ concentration up to 0.1 mM and at about 10 IlM Ca 2+ was half the maximum rate Ca2+ at concentrations over 0.1 mM, in contrast, were dosedependently inhibitory to CICR ATP, ADP, andAMPOPCP, an unhydrolyzableATP analogue, are more potent than AMP in their stimulating effect [39] Caffeine at mM increased both the Ca 2+ sensitivity in CICR and the maximum rate at the optimal Ca 2+ concentration (squares in Fig 1) However, enhancement of the maximum rate by caffeine was less than that by AMP (squares) or by other adenine nucleotides [38] The effects of caffeine and AMP are strongly potentiating to each other (filled circles) Procaine decreased the rate of CICR over the whole range of Ca2+ concentrations without significant change in Ca2+ dependency [38,40], while Mg 2+ decreased both the Ca2+ sensitivity and the maximum rate at the optimum Ca2+ concentration [38, 40] The effect ofMg2+ may be explained by the combined effect of competitive antagonistic action in stimulatory Ca2+ sites and synergistic action in an inhibitory Ca2+ concentration range Procaine and 1- rI·h-', .,. ,r-: '"7T" T"""""1 ~ at 4'C 3.0 1=0.16 o