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.     

Alpha2delta1 dihydropyridine receptor subunit is a critical element for excitation-coupled calcium entry but not for formation of tetrads in skeletal myotubes

It has been shown that small interfering RNA (siRNA) partial knockdown of the α₂δ₁ dihydropyridine receptor subunits cause a significant increase in the rate of activation of the L-type Ca²⁺ current in myotubes but have little or no effect on skeletal excitation-contraction coupling. This study used permanent siRNA knockdown of α₂δ₁ to address two important unaddressed questions. First, does the α₂δ₁ subunit contribute to the size and/or spacing of tetradic particles? Second, is the α₂δ₁ subunit important for excitation-coupled calcium entry? We found that the size and spacing of tetradic particles is unaffected by siRNA knockdown of α₂δ₁, indicating that the visible particle represents the α_1s subunit. Strikingly, >97% knockdown of α₂δ₁ leads to a complete loss of excitation-coupled calcium entry during KCl depolarization and a more rapid decay of Ca²⁺ transients during bouts of repetitive electrical stimulation like those occurring during normal muscle activation in vivo. Thus, we conclude that the α₂δ₁ dihydropyridine receptor subunit is physiologically necessary for sustaining Ca²⁺ transients in response to prolonged depolarization or repeated trains of action potentials.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

Charge movement and Ca2+ release in normal and dysgenic foetal myotubes.

Intramembrane charge movement and Ca2+ release from sarcoplasmic reticulum was studied in foetal skeletal muscle cells from normal and mutant mice with 'muscular dysgenesis' (mdg/mdg). It was shown that: 1) unlike normal myotubes, in dysgenic myotubes membrane depolarization did not evoke calcium release from the sarcoplasmic reticulum; 2) when all ionic currents are pharmacologically suppressed, membrane depolarization produced an asymmetric intramembrane charge movement in both normal and dysgenic myotubes. The relationship between the membrane potential and the amount of charge movement in these muscles could be expressed by a two-state Boltzmann equation; 3) the maximum amount of charge movement associated with depolarization (Qon max) in normal and in dysgenic myotubes was 6.3 +/- 1.4 nC/microF (n = 6) and 1.7 +/- 0.3 nC/microF (n = 6) respectively; 4) nifedipine (1-20 microM) applied to the bath reduced Qon max by about 40% in normal muscle cells. In contrast, the drug had no significant effect on the charge movement of dysgenic myotubes; and 5) the amount of nifedipine-resistant charge movement in normal and in dysgenic myotubes was 3.5 nC/microF (n = 3) and 1.7 nC/microF 1 maximum (n = 3), respectively.     

Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads

Summary Isolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetectedM _r 95 K protein which is distinct from the SR Ca²⁺ pump and phosphorylaseb. TheM _r 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified strong triads (accompanying paper). From these findings, we propose a new model for the triad junction.     

Functional interaction of CaV channel isoforms with ryanodine receptors studied in dysgenic myotubes

The L-type Ca(2+) channels Ca(V)1.1 (alpha(1S)) and Ca(V)1.2 (alpha(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (alpha(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the alpha(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for alpha(1C) and alpha(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of alpha(1C) and alpha(1A) resembled those in alpha(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. alpha(1C) but not alpha(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 microM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in alpha(1A)-expressing myotubes. In cells expressing an alpha(1A)-derivative (alpha(1)Aas(1592-clip)) containing the putative targeting sequence of alpha(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in alpha(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density.     

Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients of mouse dihydropyridine receptor (DHPR) beta 1-null myotubes expressing a mouse DHPR beta 1 cDNA have been characterized. In beta 1-null myotubes maintained in culture for 10-15 days, the density of the L-type current was approximately 7-fold lower than in normal cells of the same age (Imax was 0.65 +/- 0.05 pA/pF in mutant versus 4.5 +/- 0.8 pA/pF in normal), activation of the L-type current was significantly faster (tau activation at +40 mV was 28 +/- 7 ms in mutant versus 57 +/- 8 ms in normal), charge movements were approximately 2.5-fold lower (Qmax was 2.5 +/- 0.2 nC/microF in mutant versus 6.3 +/- 0.7 nC/microF in normal), Ca2+ transients were not elicited by depolarization, and spontaneous or evoked contractions were absent. Transfection of beta 1-null cells by lipofection with beta 1 cDNA reestablished spontaneous or evoked contractions in approximately 10% of cells after 6 days and approximately 30% of cells after 13 days. In contracting beta 1-transfected myotubes there was a complete recovery of the L-type current density (Imax was 4 +/- 0.9 pA/pF), the kinetics of activation (tau activation at +40 mV was 64 +/- 5 ms), the magnitude of charge movements (Qmax was 6.7 +/- 0.4 nC/microF), and the amplitude and voltage dependence of Ca2+ transients evoked by depolarizations. Ca2+ transients of transfected cells were unaltered by the removal of external Ca2+ or by the block of the L-type Ca2+ current, demonstrating that a skeletal-type excitation-contraction coupling was restored. The recovery of the normal skeletal muscle phenotype in beta 1-transfected beta-null myotubes shows that the beta 1 subunit is essential for the functional expression of the DHPR complex.     

Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor beta1a subunit.

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.     

Separation of dihydropyridine binding sites from cardiac junctional sarcoplasmic reticulum

When microsomes from feline ventricular muscle are centrifuged on continuous linear sucrose gradients, the major peak for the distribution pattern of the dihydropyridine binding sites corresponds in position and shape with the distribution of the Mr 300K polypeptide marker for junctional sarcoplasmic reticulum (SR). Plasma membrane vesicles are also present in those gradient fractions and appear to be joined to the junctional SR as native dyads. We now report that when such putative dyads are passed through the French press, both the dihydropyridine binding sites and the plasma membrane marker band together at a new isopycnic point distinct from the junctional SR. We conclude that as has been found in the skeletal muscle system the dihydropyridine binding sites are a marker for the junctional domain of the plasma membrane and that separation of the dyad components of the mammalian myocardium can be attained.     

Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading.

Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle.     

Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle

The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.     

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes

Skeletal and cardiac dihydropyridine receptors function both as voltage- dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L- channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L- channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.     

Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes

We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.     

Complex interactions between skeletal muscle ryanodine receptor and dihydropyridine receptor proteins.

Evidence for functional interactions between the Ca2+ release channel in the skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) and the L-type Ca2+ channel in the sarcolemma (the dihydropyridine receptor), leading to excitation-contraction coupling, is reviewed and experimental systems used to identify candidate sites of interaction are outlined.     

Influence of pHo on calcium channel block by amlodipine, a charged dihydropyridine compound. Implications for location of the dihydropyridine receptor

We have investigated the modulation of L-type calcium channel currents in isolated ventricular cells by the dihydropyridine derivative amlodipine, a weak base with a pKa of 8.6. Under conditions that favor neutral drug molecules, amlodipine block resembles other, previously described, neutral dihydropyridine derivatives: block is more pronounced at depolarized voltages, repetitive pulsing is not needed to promote block, and recovery is complete at hyperpolarized voltages. When the drug is ionized, depolarized voltages still enhance block, however, the time course is slow and speeded by repetitive pulses that open channels. Recovery from block by ionized drug molecules is very slow and incomplete, but can be rapidly modified by changes in external hydrogen ion concentration. We conclude from these observations that the degree of ionization of the drug molecule can affect access to the dihydropyridine receptor and that external protons can interact with the drug-receptor complex even if channels are blocked and closed. These observations place limitations on the location of this receptor in the ventricular cell membrane.     

Genetic effects observed in tetrads of Tradescantia induced by radon.

Inflorescences of Tradescantia clone 4430 were exposed to different concentrations of radon (Rn) gas (0.85, 12.10, 36.50 and 98.16 kBq/m3) from plants placed in an acrylic chamber that received radon from a container with pitchblende (containing uranium mineral). The exposure time was 24 h, afterwards the plants were left for 6 h in water and constantly aerated. Positive control plants were irradiated with gamma rays (0.8 Gy) and negative control plants received ambient air only (the background measurement had a mean of 0.38 kBq/m3). Micronuclei (MCN) in the tetrads induced by alpha particles emitted from Rn were tabulated and a linear concentration response was obtained. The potency of radon to induce MCN from the slope of the regression line was 0.13 MCN/kBq/m3 of alpha-radiation. Radon could reach the anthers by diffusion through the aerial spaces within the buds.     

Neuregulin inhibits acetylcholine receptor aggregation in myotubes

The high local concentration of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction results from their aggregation by the agrin/MuSK signaling pathway and their synthetic up-regulation by the neuregulin/ErbB pathway. Here, we show a novel role for the neuregulin/ErbB pathway, the inhibition of AChR aggregation on the muscle surface. Treatment of C2C12 myotubes with the neuregulin epidermal growth factor domain decreased the number of both spontaneous and agrin-induced AChR clusters, in part by increasing the rate of cluster disassembly. Upon cluster disassembly, AChRs were internalized into caveolae (as identified by caveolin-3). Time-lapse microscopy revealed that individual AChR clusters fragmented into puncta, and application of neuregulin accelerated the rate at which AChR clusters decreased in area without affecting the density of AChRs remaining in individual clusters (as measured by the fluorescence intensity/unit area). We propose that this novel action of neuregulin regulates synaptic competition at the developing neuromuscular junction.     

Regulation of acetylcholine receptor desensitization in mouse myotubes by cytosolic cyclic AMP

Whole-cell currents activated by bath applications of acetylcholine (ACh) (10-30 microM) were recorded from patch-clamped myotubes of the mouse C2 cell line. Increasing concentrations of forskolin caused a dose-dependent fast decay of ACh-activated currents as compared to the long-lasting ACh-currents in control cells. The forskolin-induced modulation of nicotinic ACh receptor (nAChR) desensitization was proportional to the drug-induced elevation in the cyclic AMP (cAMP) cellular content. Furthermore, an increase in the rate of decay of the ACh-current response, which paralleled an elevation in cAMP cellular content, was caused by treatment with a calcitonin gene-related peptide (1 microM), 8-Br-cAMP (0.5 mM), or by loading the myotubes with cAMP. These results therefore indicate that the desensitization of nAChR is a cAMP-related process in C2-myotubes.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.