Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.     

Ca2+ signaling in microdomains: Homer1 mediates the interaction between RyR2 and Cav1.2 to regulate excitation-contraction coupling

Excitation-contraction (E-C) coupling and Ca(2+)-induced Ca(2+) release in smooth and cardiac muscles is mediated by the L-type Ca(2+) channel isoform Ca(v)1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca(v)1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca(v)1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1(-/-) mice and by demonstrating a Homer1-mediated dynamic interaction between Ca(v)1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca(v)1.2. Most notably, expression of Ca(v)1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca(v)1.2 and RyR2 to regulate the intensity of Ca(2+)-induced Ca(2+) release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca(v)1.2 and RyR2. In one state, Ca(v)1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca(v)1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca(v)1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.     

Hypertension-induced remodeling of cardiac excitation-contraction coupling in ventricular myocytes occurs prior to hypertrophy development

Hypertension is a major risk factor for developing cardiac hypertrophy and heart failure. Previous studies show that hypertrophied and failing hearts display alterations in excitation-contraction (E-C) coupling. However, it is unclear whether remodeling of the E-C coupling system occurs before or after heart disease development. We hypothesized that hypertension might cause changes in the E-C coupling system that, in turn, induce hypertrophy. Here we tested this hypothesis by utilizing the progressive development of hypertensive heart disease in the spontaneously hypertensive rat (SHR) to identify a window period when SHR had just developed hypertension but had not yet developed hypertrophy. We found the following major changes in cardiac E-C coupling during this window period. 1) Using echocardiography and hemodynamics measurements, we found a decrease of left ventricular ejection fraction and cardiac output after the onset of hypertension. 2) Studies in isolated ventricular myocytes showed that myocardial contraction was also enhanced at the same time. 3) The action potential became prolonged. 4) The E-C coupling gain was increased. 5) The systolic Ca(2+) transient was augmented. These data show that profound changes in E-C coupling already occur at the onset of hypertension and precede hypertrophy development. Prolonged action potential and increased E-C coupling gain synergistically increase the Ca(2+) transient. Functionally, augmented Ca(2+) transient causes enhancement of myocardial contraction that can partially compensate for the greater workload to maintain cardiac output. The increased Ca(2+) signaling cascade as a molecular mechanism linking hypertension to cardiac hypertrophy development is also discussed.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.     

Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro

Muscular dysgenesis (mdg) in mice causes the failure of excitation-contraction (E-C) coupling in skeletal muscle. Cultured dysgenic muscle fails to contract upon depolarization, lacks typical muscle ultrastructure, including normal triads, and lacks functional voltage-dependent slow calcium channels. We show that normal rodent fibroblasts and 3T3 fibroblasts "rescue" dysgenic myotubes, reestablishing contractions (i.e., E-C coupling), normal ultrastructure, and functional slow calcium channels. These results support the finding that the expression of the slow calcium channel is affected in the mdg mutation and that this protein is essential for E-C coupling. Additionally, fibroblast rescue provides a system for examining the mechanisms of heterotypic cellular influence on cell function.     

Induction of normal ultrastructure by CGRP treatment in dysgenic myotubes

The calcitonin gene-related peptide (CGRP) restores an apparent normal ultrastructure in mdg/mdg muscle cells in vitro, including a normal triadic organization which is known to be essential for excitation-contraction (E-C) coupling. However, neither slow L-type Ca2+ channel activity nor E-C coupling, which are absent in mdg/mdg muscle, were re-established. These observations suggest a potential role of CGRP (and also of cAMP as the intracellular messenger) in the morphological development of the muscle fiber.     

Discoupling of excitation-contraction processes by urea treatment of frog skeletal muscle

On exposure (E) of frog semitendinosus muscle to 400 mmol/l urea (U) in sodium chloride Ringer's solution, the tension development to isoK+ solutions decreased, while in choline chloride Ringer it increased. On quick removal (R) of urea, always a block of excitation-contraction (E-C) coupling occurred accompanied by transient or persistent swelling of fibres and a similar but definite decrease of their resting membrane potential (Fig. 2). Muscle contraction could be elicited by caffeine even after UER-treatment but then only the slow tension increase (second phase of normal caffeine contraction) occurred (Fig. 3a). The fast tension increase to caffeine (first phase) could be restored if after UER-treatment 5 mmol/l mannitol (Fig. 3b), a 20 min treatment with choline chloride (Fig. 4a) or sodium isethionate (Fig. 4b) Ringer's solution of double osmolarity were applied. Caffeine contraction could not be elicited when sodium chloride Ringer's solution of double osmolarity was used under similar conditions (Fig. 5). E-C block to isoK+ solution persisted in all these experiments. E-C coupling could partially be restored by short treatment of muscle with caffeine (Figs 6a, b).     

Surface plasmon resonance spectroscopy in the study of membrane-mediated cell signalling.

Peptide-membrane interactions contribute to many important biological processes such as cellular signaling, protein trafficking and ion-channel formation. During receptor-mediated signalling, activated intracellular signalling molecules are often recruited into receptor-induced signaling complexes at the cytoplasmic surface of the cell membrane. Such recruitment can depend upon protein-protein and protein-lipid interactions as well as protein acylation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study these interactions and have provided important information on the relationship between the structure of these proteins involved in cell signalling and their biological function. More recently, surface plasmon resonance (SPR) spectroscopy has also been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications, which demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated cellular signalling.     

Surface plasmon resonance spectroscopy: an emerging tool for the study of peptide-membrane interactions

The interactions between peptides and membranes mediate a wide variety of biological processes, and characterization of the molecular details of these interactions is central to our understanding of cellular events such as protein trafficking, cellular signaling and ion-channel formation. A wide variety of biophysical techniques have been combined with the use of model membrane systems to study peptide-membrane interactions, and have provided important information on the relationship between membrane-active peptide structure and their biological function. However, what has generally not been reported is a detailed analysis of the affinity of peptide for different membrane systems, which has largely been due to the difficulty in obtaining this information. To address this issue, surface plasmon resonance (SPR) spectroscopy has recently been applied to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. This article provides an overview of these recent applications that demonstrate the potential of SPR to enhance our molecular understanding of membrane-mediated peptide function.     

Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling

Muscular dysgenesis (mdg) is a lethal autosomal, recessive mutation of mice. Skeletal muscle from dysgenic mice is paralyzed due to the failure of excitation-contraction (E-C) coupling. Considerable evidence indicates that this failure results from the absence of a specific gene product, the alpha 1 subunit of the skeletal muscle receptor for dihydropyridine calcium channel modifiers. This dihydropyridine receptor is hypothesized to function in E-C coupling of normal skeletal muscle as the voltage sensor that triggers calcium release from the sarcoplasmic reticulum and thereby causes contraction. The skeletal muscle dihydropyridine receptor is also postulated to function as the ion channel responsible for a slowly activating, dihydropyridine-sensitive calcium current (Islow). Dysgenic skeletal muscle lacks Islow but expresses, at low levels, a distinctly different dihydropyridine-sensitive calcium current (Idys). The channel protein underlying Idys is incapable of serving as a voltage sensor for E-C coupling. Studies using dysgenic skeletal muscle have provided significant insight into the role of dihydropyridine receptors in E-C coupling.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of muscarinic stimulation and mode of excitation-contraction coupling in bovine trachealis muscle

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K+ channel blocker, nifedipine almost completely inhibited contractions induced by the M3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M3 receptor is coupled to a cellular pathway linked with the activation of K+ channels that exerts a potent functional antagonism against activation of voltage-dependent Ca2+ entry.     

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.     

Cell-cell signaling via Eph receptors and ephrins

Eph receptors are the largest subfamily of receptor tyrosine kinases regulating cell shape, movements, and attachment. The interactions of the Ephs with their ephrin ligands are restricted to the sites of cell-cell contact since both molecules are membrane attached. This review summarizes recent advances in our understanding of the molecular mechanisms underlining the diverse functions of the molecules during development and in the adult organism. The unique properties of this signaling system that are of highest interest and have been the focus of intense investigations are as follows: (i) the signal is simultaneously transduced in both ligand-expressing cells and receptor-expressing cells, (ii) signaling via the same molecules can generate opposing cellular reactions depending on the context, and (iii) the Ephs and the ephrins are divided into two subclasses with promiscuous intrasubclass interactions, but rarely observed intersubclass interactions.     

Diffusion-based determination of protein homodimerization on reconstituted membrane surfaces

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions—the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton’s tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions.     

Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres

Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.     

Structure of the reovirus membrane-penetration protein, Mu1, in a complex with is protector protein, Sigma3

Cell entry by nonenveloped animal viruses requires membrane penetration without membrane fusion. The reovirus penetration agent is the outer-capsid protein, Mu1. The structure of Mu1, complexed with its "protector" protein, Sigma3, and the fit of this Mu1(3)Sigma3(3) heterohexameric complex into the cryoEM image of an intact virion, reveal molecular events essential for viral penetration. Autolytic cleavage divides Mu1 into myristoylated Mu1N and Mu1C. A long hydrophobic pocket can receive the myristoyl group. Dissociation of Mu1N, linked to a major conformational change of the entire Mu1 trimer, must precede myristoyl-group insertion into the cellular membrane. A myristoyl switch, coupling exposure of the fatty acid chain, autolytic cleavage of Mu1N, and long-range molecular rearrangement of Mu1C, thus appears to be part of the penetration mechanism.     

Magnetic fields,radicals and cellular activity

Some effects of low-intensity magnetic fields on the concentration of radicals and their influence on cellular functions are reviewed. These fields have been implicated as a potential modulator of radical recombination rates. Experimental evidence has revealed a tight coupling between cellular function and radical pair chemistry from signaling pathways to damaging oxidative processes. The effects of externally applied magnetic fields on biological systems have been extensively studied, and the observed effects lack sufficient mechanistic understanding. Radical pair chemistry offers a reasonable explanation for some of the molecular effects of low-intensity magnetic fields, and changes in radical concentrations have been observed to modulate specific cellular functions. Applied external magnetic fields have been shown to induce observable cellular changes such as both inhibiting and accelerating cell growth. These and other mechanisms, such as cell membrane potential modulation, are of great interest in cancer research due to the variations between healthy and deleterious cells. Radical concentrations demonstrate similar variations and are indicative of a possible causal relationship. Radicals, therefore, present a possible mechanism for the modulation of cellular functions such as growth or regression by means of applied external magnetic fields.