Copyright ©ERS Journals Ltd 1998 Eur Respir J 1998; 11: 758–766 European Respiratory Journal DOI: 10.1183/09031936.98.11030758 ISSN 0903 - 1936 Printed in UK - all rights reserved SERIES 'CELL BIOLOGY OF RESPIRATORY MUSCLES' Edited by M. Decramer and M. Aubier Number 4 in this Series Calcium ATPase and respiratory muscle function M. Aubier, N.Viires aa Calcium ATPase and respiraory muscle function. M. Aubier, N. Viires. ©ERS Journals Ltd INSERM U 408, Unité de Pneumologie, 1998. Hôpital Bichat, 46, rue H. Huchard, 75018 ABSTRACT: The sarcoplasmic reticulum (SR) of striated muscle is a highly special- Paris, France. ized intracellular membrane system that plays a key role in the contraction-relaxa- tion cycle of muscle. Its primary function is the regulation of cytoplasmic Ca2+ Correspondence: M. Aubier concentration. A key element in this regulation is the Sarco(endo)plasmic reticulum Unité de Pneumologie 2+ 2+ 46, rue Henri Huchard Ca -adenosine triphosphatase (SERCA), which by sequestering Ca into the SR, 75018 Paris induces and maintains relaxation. It has been extensively studied with respect to France structure and mechanism of action, and more recently to gene expression. Three sep- Fax: 33 140258818 arate genes encode five SERCA isoforms, two of which, SERCA 1 and SERCA 2, are expressed in skeletal muscle. Keywords: Diaphragm In the first part of this review we focus on the general properties of the Ca2+ pump respiratory muslces (structure and function and regulation of activity). In the second part we describe sarcoplasmic reticulum variations in SERCA expression in various physiological and pathological situations. SERCA pumps These have essentially been studied in the heart and skeletal muscles, with data in res- piratory muscles being very limited. Received: September 29 1997 Accepted after revision November 15 1997 Eur Respir J 1998; 11: 758–766. The major proteins responsible for contraction and relax- sarcolemma through transverse tubules (T tubules). Depo- ation in skeletal muscle are myosin and the sarcoplasmic larizing currents in the transverse tubule culminate in a reticulum (SR) Ca2+-adenosine triphosphatase (ATPase), signal for Ca2+ release from the SR, which in turn initiates respectively. Both these proteins exist as multiple isoforms muscle contraction. The SR has two additional functions and contribute to defining skeletal muscle phenotype. essential to excitation-contraction coupling, namely Ca2+ While changes in myosin isoform composition have been reuptake to initiate muscle relaxation, and Ca2+ storage to extensively studied in physiopathological situations, com- maintain relaxed muscle in a quiescent state. The ability paratively little is known of the expression or regulation of of this system to regulate cytoplasmic Ca2+ concentrations the Ca2+-ATPase isoforms. plays a central role in the contraction-relaxation cycle of Ca2+-ATPases constitute a large family of proteins that skeletal, cardiac and, to a lesser degree, smooth muscle fall into two distinct groups, the sarco(endo)plasmic retic- [1–5]. ulum Ca2+-ATPase (SERCA), and the plasma membrane In recent years, an understanding of the molecular Ca2+-ATPase (PMCA). Most eukaryotic cells coexpress, events involved in Ca2+ regulation by the SR has come in a tissue-specific and differentiation stage-specific man- about through resolution of the sarco-tubular system into ner, one or more types of SERCA and PMCA pumps. This its component membrane domains and through isolation, review will focus on the SERCA pumps. reconstitution and biochemical analysis of individual pro- The skeletal muscle SR Ca2+-ATPase is part of the teins in these domains. SERCA family of calcium pumps involved in the trans- In skeletal muscles, the SR membrane system in situ port of calcium from the cytosol to various intracellular is composed of two distinct portions: 1) voluminous, mat- stores such as the SR, the endoplasmic reticulum (ER) and rix filled terminal cisternae which are associated with calciosomes. It is present in several cell types and plays an the transverse tubule; and 2) the longitudinal SR,which important role in controlling cellular functions such as contains very little lumenal structure and connects medi- relaxation and secretion. In skeletal muscle it is localized ally with the two terminal cisternae [6]. in the SR. It is now clear that certain functions of the SR are restrict- ed to specific regions of this membrane system [7] (fig. 1). Sarcoplasmic reticulum Early fractionation of the SR by sucrose gradient cen- trifugation and subsequently freeze fracture techniques, The SR is an intracellular membrane network that is showed that two distinct heavy and light fractions could in close contact with the myofibrils and couples with the be isolated [6, 8]. Previous articles in this series: No. 1: G.C. Sieck, Y.S. Prakash. Cross bridge kinetics in respiratory muscles. Eur Respir J 1997; 10: 2147–2158. No. 2: J.G. Gea. Myosin gene expression in the respiratory muscles. Eur Respir J 1997; 10: 2404–2410. No. 3: B.J. Petrof. Respiratory muscles as a target for adenovirus-mediated gene therapy. Eur Respir J 1998; 11: 492–497. CALCIUM ATPASE AND RESPIRATORY MUSCLE FUNCTION 759 Sarcolemma Nucleotide t tubule PLN binding domain Terminal cisternae + β-domain * p Longitudinal sarcoplasmic reticulum + * * + Cytoplasmic domain Phospholamban 2+ Ryanodine Ca -ATPase receptor NH2 Fig. 1. – Schematic representation of the sarcoplasmic reticulum show- Stalk COOH ing the arrangement of constituent proteins. Within the longitudinal domain membrane the major protein is the Ca2+-adenosine triphosphatase (ATPase). Phospholamban is also present with a similar distribution to Transmembrane the Ca2+ pump. The terminal cisternae contains the acidic calcium bind- domain ing proteins calsequestrin (●), calreticulin (+) and a 170 kDa protein, now referred to as the histidine rich Ca2+ binding protein (*). M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 Fig. 2. – Structural diagram of the Ca2+-adenosine triphosphatase The light fraction, which corresponds predominantly to (ATPase) molecule. Ten putative transmembrane segments (M1–M10) are shown. Sites for the binding of phospholamban (PLN) and for regu- the longitudinal SR, is primarily concerned with the up- 2+ 2+ latory serine phosphorylation of the sarco(endo)plasmic reticulum Ca take of calcium and contains the 110 kDa Ca -ATPase as ATPase (SERCA) 2 isoform (P) are also indicated. Modified from [1, it's major contituent. Lumenal glycoproteins of 53 and 24]. 160 kDa are also present in this fraction. In cardiac mus- cle and in slow twitch skeletal muscle, the regulatory pro- 2+ tein phospholamban (a homomeric pentamer of 6 kDa the cytoplasm. It catalyses Ca transport to the lumen of subunits), which is thought to interact with the calcium the SR by an active process that requires adenosine tripho- pump and mediate the effects of catecholamines on Ca2+ sphate (ATP). Enzyme phosphorylation and ATP hydroly- 2+ transport, is present with a similar distribution to the Ca2+ sis result in translocation of the two Ca ions bound to the pump. enzyme from a high affinity site to a low affinity site. The The heavy fraction, corresponding to the terminal cis- two calcium ions are then released into the lumen of the ternae, is the site of calcium release and storage. It con- SR (fig. 3). tains the calcium release channel or ryanodine receptor, The Ca2+-ATPase has been purified and its primary which is a high molecular weight tetramer made up of structure determined by direct amino acid sequence deter- 565 kDa subunits. The acidic calcium binding proteins mination and by complementary deoxyribonucleic acid calsequestrin, calreticulin and a 170 kDa protein now re- (DNA) cloning (fig. 2). Analysis of hydrophobic sequen- ferred to as the histidine rich Ca2+ binding protein, are also ces led to the assignment of 10 transmembrane helices located in this fraction [9–12]. (M1 to M10): four in the NH2 terminal quarter and six in Recent molecular cloning analysis have demonstrated the existence of distinct isoforms of many of these pro- teins. Skeletal muscle isoforms of the ryanodine receptor, High affinity Low affinity the calcium ATPase (SERCA) and calsequestrin have Cytoplasm been identified, although very little is known about the 1 inter-relations between these isoforms or about their regu- 2 2 1 lation. 4 5 Uptake 3 4 5 Stalk 3 Structure and function 10 9 7 8 6 7 8 6 9 The SERCA of the SR plays a key role in regulation of Ca2+ skeletal muscle function. By pumping calcium from the Ca2+ sarcoplasm to luminal spaces in the organelle it lowers Membrane sarcoplasmic Ca2+ concentration (to the range of 100 nM), thereby inducing and maintaining muscle relaxation. It re- presents 60–80% of the total protein content in the SR of Lumen adult animals and has been extensively studied with res- pect to its structure, reaction kinetics and gene expression Discharge Fig. 3. – Model illustrating the mechanism of Ca2+ transport by the [13–25]. 2+ 2+ Ca -adenosine triphosphatase (ATPase). In the high affinity state, high The Ca -ATPase is a single large polypeptide with a affinity Ca2+ binding sites located near the centre of the transmembrane molecular weight of 100 kDa. Electron microscopic and domain are accessible to cytoplasmic calcium but not to luminal cal- x-ray diffraction studies have revealed that it is comp- cium. The sites are made up from amino acid residues located in pro- rised of a cytoplasmic headpiece and stalk sectors and a posed transmembrane sequences M4, M5, M6 and M8. Conformational changes induced by adenosine triphosphate (ATP) hydrolysis lead to the transmembrane basepiece, making up a tripartite structure low affinity state, in which high affinity calcium binding sites are dis- (fig. 2). The enzyme is asymetrically oriented in the mem- rupted, access to the sites by cytoplasmic Ca2+ is closed off and access brane with virtually all of its extramembranous mass in to the sites by luminal calcium is gained.