Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse

Human X-linked Duchenne and Becker muscular dystrophies are due to defects in dystrophin, the product of an exceptionally large gene. Although dystrophin has been characterized as a spectrin-like submembranous cytoskeletal protein, there is no experimental evidence for its function in the structural maintenance of muscle. Current hypotheses attribute necrosis of dystrophin-less fibres in situ to mechanical weakening of the outer membrane, to an excessive influx of Ca2+ ions, or to a combination of these two mechanism, possibly mediated by stretch-sensitive ion channels. Using hypo-osmotic shock to determine stress resistance and a mouse model (mdx) for the human disease, we show that functional dystrophin contributes to the stability of both cultured myotubes and isolated mature muscle fibres.     

Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane

It has been known for some years that skeletal muscle develops a high potassium permeability in conditions that produce rigor, where ATP concentrations are low and intracellular Ca2+ is high. It has seemed natural to attribute this high permeability to K channels that are opened by internal Ca2+, especially as the presence of such channels has been demonstrated in myotubes and in the transverse tubular membrane system of adult skeletal muscle. However, as we show here, the surface membrane of frog muscle contains potassium channels that open at low internal concentrations of ATP (less than 2 mM). ATP induces closing of these channels without being split, apparently holding the channels in one of a number of closed states. The channels have at least two open states whose dwell times are voltage-dependent. Surprisingly, we find that these may be the most common K channels of the surface membrane of skeletal muscle.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.     

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.     

Restoration of dysgenic muscle contraction and calcium channel function by co-culture with normal spinal cord neurons

Muscular dysgenesis (mdg) is a spontaneous recessive lethal mutation in the mouse. The disease is characterized by a total lack of excitation-contraction coupling in embryonic skeletal muscle. This developmental abnormality is associated with a drastic deficiency in the expression of voltage-sensitive Ca2+ channels in skeletal muscle without alteration of the properties of voltage-sensitive Na+ channels or of voltage-sensitive Ca2+ channels in cardiac and neuronal cells. Membrane couplings between sarcoplasmic reticulum and the transverse tubules, known as triads, were also found to be drastically altered in embryonic muscle of the homozygous mutant (mdg/mdg). Triads in the mdg/mdg muscle were less numerous, disorganized and lacked spaced densities. This paper shows that co-culture of mdg/mdg myotubes with normal spinal cord neurons re-establishes Ca2+ channel activity, contraction and normal triad organization. The decrease thus cannot be due to a mutation of the Ca2+ channel as previously suggested. Normal nerve cells may supply an essential factor to mutant muscle cells.     

Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle.

Dystrophin is associated with a complex of muscle membrane (sarcolemmal) glycoproteins that provide a linkage to the extracellular matrix protein, laminin. The absence of dystrophin leads to a dramatic reduction of the dystrophin-associated proteins (156DAG, 59DAP, 50DAG, 43DAG and 35DAG) in the sarcolemma of patients with Duchenne muscular dystrophy and mdx mice. Here we demonstrate that dystrophin-related protein (DRP, utrophin), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins and that DRP and the dystrophin/DRP-associated proteins colocalize to the neuromuscular junction in Duchenne muscular dystrophy and mdx muscle. The DRP and dystrophin/DRP-associated proteins are found throughout the sarcolemma in small-calibre skeletal muscles and cardiac muscle of adult mdx mice. Because these muscles show minimal pathological changes, our results could provide a basis for the upregulation of DRP as a potential therapeutic approach.     

Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling

It is thought that in skeletal muscle excitation-contraction (EC) coupling, the release of Ca2+ from the sarcoplasmic reticulum is controlled by the dihydropyridine (DHP) receptor in the transverse tubular membrane, where it serves as the voltage sensor. We have shown previously that injection of an expression plasmid carrying the skeletal muscle DHP receptor complementary DNA restores EC coupling and L-type calcium current that are missing in skeletal muscle myotubes from mutant mice with muscular dysgenesis. This restored coupling resembles normal skeletal muscle EC coupling, which does not require entry of extracellular Ca2+. By contrast, injection into dysgenic myotubes of an expression plasmid carrying the cardiac DHP receptor cDNA produces L-type calcium current and cardiac-type EC coupling, which does require entry of extracellular Ca2+. To identify the regions responsible for this important functional difference between the two structurally similar DHP receptors, we have expressed various chimaeric DHP receptor cDNAs in dysgenic myotubes. The results obtained indicate that the putative cytoplasmic region between repeats II and III of the skeletal muscle DHP receptor is an important determinant of skeletal-type EC coupling.     

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts

An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.     

Repeat I of the dihydropyridine receptor is critical in determining calcium channel activation kinetics.

Membrane depolarization causes many kinds of ion channels to open, a process termed activation. For both Na+ channels and Ca2+ channels, kinetic analysis of current has suggested that during activation the channel undergoes several conformational changes before reaching the open state. Structurally, these channels share a common motif: the central element is a large polypeptide with four repeating units of homology (repeats I-IV), each containing a voltage-sensing region, the S4 segment. This suggests that the distinct conformational transitions inferred from kinetic analysis may be equated with conformational changes of the individual structural repeats. To investigate the molecular basis of channel activation, we constructed complementary DNAs encoding chimaeric Ca2+ channels in which one or more of the four repeats of the skeletal muscle dihydropyridine receptor are replaced by the corresponding repeats derived from the cardiac dihydropyridine receptor. We report here that repeat I determines whether the chimaeric Ca2+ channel shows slow (skeletal muscle-like) or rapid (cardiac-like) activation.     

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.     

Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity.

During formation of the neuromuscular junction, acetylcholine receptors in the endplate membrane become metabolically stabilized under neural control, their half-life increasing from about 1 day to about 10 days. The metabolic stability of the receptors is regulated by the electrical activity induced in the muscle by innervation. We report here that metabolic stabilization of endplate receptors but not of extrajunctional receptors can be induced in the absence of muscle activity if muscles are treated with the calcium ionophore A23187. Acetylcholine receptor stabilization was also induced by culturing non-stimulated muscle in elevated K+ with the Ca2+ channel activator (+)-SDZ202-791. Conversely, activity-dependent receptor stabilization is prevented in muscle stimulated in the presence of the Ca2+ channel blockers (+)-PN200-110 or D-600. Treatment of muscles with ryanodine, which induces Ca2+ release from the sarcoplasmic reticulum in the absence of activity, does not cause stabilization of junctional receptors. Evidently, muscle activity induces metabolic acetylcholine receptor stabilization by way of an influx of Ca2+ ions through dihydropyridine-sensitive Ca2+ channels in the endplate membrane, whereas Ca2+ released from the sarcoplasmic reticulum is ineffective in this developmental process.     

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.     

Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes

Hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)-P2) to water soluble inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a common response by many different kinds of cells to a wide variety of external stimuli (see refs 1 and 2 for review). Ins (1,4,5)P3 is a putative second messenger which increases intracellular Ca2+ by mobilizing internal Ca2+ stores, a hypothesis which has been substantiated by studies with chemically permeabilized cells and with isolated microsomal membrane fractions. But the possibility that Ins(1,4,5)P3 could induce in intact cells an influx of external Ca2+ through transmembrane channels, originally hypothesized by Michell in 1975, has never been directly tested. We report here single-channel recordings of an Ins(1,4,5)P3-activated conductance in excised patches of T-lymphocyte plasma membrane. The Ins(1,4,5)P3-activated transmembrane channel appears to be identical to the recently described mitogen-regulated, voltage-insensitive Ca2+ permeable channel involved in T-cell activation. We suggest that Ins(1,4,5)P3 acts as the second messenger mediating transmembrane Ca2+ influx through specific Ca2+-permeable channels in mitogen-stimulated T-cell activation.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs

Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Crystal structure and mechanism of a calcium-gated potassium channel

Ion channels exhibit two essential biophysical properties; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.     

Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.

Calcium has an important role in regulating epithelial cell ion transport and is itself transported by tissues involved in the maintenance of extracellular Ca2+ homeostasis. Although the mechanism of Ca2+ entry in electrically excitable cells is well-documented little is known about it in epithelial cells. Calcium absorption in polarized epithelial cells is a two-step process in which Ca2+ enters cells across apical plasma membranes and is extruded across basolateral membranes. Efflux may be mediated by an energy-dependent Ca2(+)-ATPase or by Na+/Ca2+ exchange. We examined Ca2+ influx in single cultured cells from distal renal tubules sensitive to parathyroid hormone by measuring intracellular Ca2+. Our results demonstrate that parathyroid hormone activates dihydropyridine-sensitive channels responsible for Ca2+ entry. We also show that microtubule-dependent exocytosis stimulated by parathyroid hormone may be necessary for the insertion or activation of Ca2+ channels in these cells. Once inserted or activated, dihydropyridine-sensitive channels mediate Ca2+ entry into these Ca2(+)-transporting epithelial cells. Our results support the view that agonist-induced exocytosis may represent a general paradigm for modulation of transport in epithelial cells by delivery and incorporation of transport proteins to plasma membranes or by delivery to plasma membranes of factors regulating these proteins.     

Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels

Animal cells initially swell in hypotonic media by osmotic water equilibration, but their volume is subsequently regulated by a net loss of KCl and amino acids with concomitant loss of cell water. Mechanisms for regulating cell volume are important in allowing cells to adapt to variations in external tonicity and metabolic load. In red cells the KCl loss is mediated by electroneutral ion transport mechanisms. In contrast, conductive K+ and Cl- transport pathways are activated during regulatory volume decrease in several cell types including epithelia. The activation seems to be mediated by internal Ca2+, but the detailed mechanism is not known. In a leaky epithelium, the choroid plexus epithelium, we have found a cation-selective, Ca2+-permeable channel which opens with membrane stretch. The epithelium also contains a high density of the large (approximately 200 pS) type of Ca2+- voltage-activated K+ channel. Both channels are normally closed. I propose that in hypotonic media, the stretching of the cell membrane produced by the initial swelling causes influx of Ca2+ through the stretch-activated channels, which activates the neighbouring large K+ channels to produce increased K+ outflux with associated loss of cell water.