Sarco(endo)plasmic reticulum calcium ATPase (SERCA) inhibition by sarcolipin is encoded in its luminal tail.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated in a tissue-dependent manner via interaction with the short integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). Although defects in SERCA activity are known to cause heart failure, the regulatory mechanisms imposed by PLN and SLN could have clinical implications for both heart and skeletal muscle diseases. PLN and SLN have significant sequence homology in their transmembrane regions, suggesting a similar mode of binding to SERCA. However, unlike PLN, SLN has a conserved C-terminal luminal tail composed of five amino acids ((27)RSYQY), which may contribute to a distinct SERCA regulatory mechanism. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg(27) and Tyr(31) are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg(27)stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg(27)stop form of SLN resulted in loss of function, whereas the PLN chimeras resulted in superinhibition with characteristics of both PLN and SLN. Based on our results, we propose that the C-terminal tail of SLN is a distinct, essential domain in the regulation of SERCA and that the functional properties of the SLN tail can be transferred to PLN.

State-dependent block of Na+ channels by articaine via the local anesthetic receptor.

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na+ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na+ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na+ channels at -140 mV with a 50% inhibitory concentration (IC(50)) of 378 +/- 26 microM (n = 5). The affinity was higher for inactivated Na+ channels measured at -70 mV with an IC50 value of 40.6 +/- 2.7 microM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na+ channels with an IC50 value of 15.8 +/- 1.5 microM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na+ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9x) > inactivated (9.3x) > resting (1x) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na+ channels, with IC(50) values of 8.8 +/- 0.1 and 22.0 +/- 0.5 microM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na+ channel isoforms.

Molecular analysis of protein assembly in muscle development.

The challenge presented by myofibril assembly in striated muscle is to understand the molecular mechanisms by which its protein components are arranged at each level of organization. Recent advances in the genetics and cell biology of muscle development have shown that in vivo assembly of the myofilaments requires a complex array of structural and associated proteins and that organization of whole sarcomeres occurs initially at the cell membrane. These studies have been complemented by in vitro analyses of the renaturation, polymerization, and three-dimensional structure of the purified proteins.

An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator.

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.

Skeletal muscle: anatomy, physiology, and pathophysiology.

Many questions remain to be answered. The intention here is to attempt to place in perspective a portion of what is known and understood scientifically, to make working sense of the pathophysiologic process so that we as clinicians can "in our mind's eye" understand what it is that we are trying to treat with what now seems to be a multitude of therapies. When a patient presents to your office with acute or chronic, or both, myofascial pain dysfunction syndrome, some basic questions must be addressed. Is the disease process genetic (intrinsic) or acquired (extrinsic), or both? It is the purpose of diagnosis to unearth the underlying predispositions that patients may exhibit. Often the predispositions are quite obvious (gross postural discrepancies and skeletal and dental malrelationships( or they may be consummately subtle (endocrinopathies and behavioral patterns). It seems that the main job to be completed is diagnosis followed by the utilization of well-known physical medicine (conservative) techniques that treat the source of the disorder: the myofascial trigger zone.

Tracking endogenous amelogenin and ameloblastin in vivo.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

Kindlin-2 controls bidirectional signaling of integrins.

Control of integrin activation is required for cell adhesion and ligand-induced signaling. Here we report that loss of the focal adhesion protein Kindlin-2 in mice results in peri-implantation lethality caused by severe detachment of the endoderm and epiblast from the basement membrane. We found that Kindlin-2-deficient cells were unable to activate their integrins and that Kindlin-2 is required for talin-induced integrin activation. Furthermore, we demonstrate that Kindlin-2 is required for integrin outside-in signaling to enable firm adhesion and spreading. Our findings provide evidence that Kindlin-2 is a novel and essential element of bidirectional integrin signaling.

The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish.

The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.

The carboxyl-terminal region of cyclic nucleotide-modulated channels is a gating ring, not a permeation path.

The recent elucidation of the structure of the carboxyl-terminal region of the hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channel has prompted us to investigate a curious feature of this structure in HCN2 channels and in the related CNGA1 cyclic nucleotide-gated (CNG) channels. The crystallized fragment of the HCN2 channel contains both the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. At the center of the fourfold-symmetric structure is a tunnel that runs perpendicular to the membrane. The narrowest part of the tunnel is approximately 10 A in diameter and is lined by a ring of negatively charged amino acids: D487, E488, and D489. Many ion channels have "charge rings" that focus permeant ions at the mouth of the pore and increase channel conductance. We used nonstationary fluctuation analysis and single-channel recording, coupled with site-directed mutagenesis and cysteine modification, to determine whether this part of HCN and CNG channels might be an extension of the permeation pathway. Our results indicate that modifying charge-ring amino acids affects gating but not ion permeation in HCN2 and CNG channels. Thus, this portion of the channel is not an obligatory part of the ion path but instead acts as a "gating ring." The carboxyl-terminal region of these channels must hang below the pore much like the "hanging gondola" of voltage-gated potassium channels, but the permeation pathway must exit the protein before the level of the ring of charged amino acids.

Cellular expression of eve1 suggests its requirement for the differentiation of the ameloblasts and for the initiation and morphogenesis of the first tooth in the zebrafish (Danio rerio).

even-skipped-related (evx) genes encode homeodomain-containing transcription factors that are involved in a series of developmental processes such as posterior body patterning and neurodifferentiation. Although evx1 and evx2 were not reported to be expressed during mammalian tooth development, we present here evidence that eve1, the closest paralog of evx1 in the actinopterygian lineage, is expressed during pharyngeal tooth formation in the zebrafish, Danio rerio. We have performed whole-mount in situ hybridization on zebrafish embryos and larvae ranging from 24 to 192 hours postfertilization (hpf). A detailed analysis of serial sections through the pharyngeal region of whole-mount hybridized and control specimens indicates that only dental epithelial cells express eve1. eve1 transcription was activated at 48 hpf, in the placode of the first tooth (i.e., the initiation site of tooth 4V(1)), and maintained in the dental epithelium throughout morphogenesis. Then, by 72 hpf, eve1 expression was restricted to the differentiating ameloblasts of the enamel organ during early differentiation stage, and this expression decreased as soon as matrix was deposited. In subsequent primary teeth (3 V(1) and 5 V(1)) as well as in their successors (replacement teeth 4V(2), 3V(2), and 5V(2)), eve1 expression was restricted to the differentiating ameloblasts and, again, disappeared when matrix was deposited. Therefore, in the zebrafish, eve1 expression in the pharyngeal region is correlated with two key steps of tooth development: initiation and morphogenesis of the first tooth, and ameloblast differentiation of all developing teeth.

Dysgenesis of cephalic neural crest derivatives in Pax7-/- mutant mice.

Pax7 is a member of the paired box containing gene family. Its expression pattern suggests a function in cephalic neural crest derivatives, skeletal muscle and central nervous system development. To understand the role of Pax7 during mouse embryogenesis, we used the homologous recombination technique in embryonic stem cells and generated Pax7-/- mice. Homozygous animals are born but die shortly afer weaning. They exhibit malformations in facial structures involving the maxilla and nose. Our analysis suggests that the observed phenotype is due to a cephalic neural crest defect. No obvious phenotype could be detected in the central nervous system and skeletal muscle. Functional redundancy between Pax7 and Pax3 is discussed.

Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase.

In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.

Giant sarcoplasmic reticulum vesicles: a study of membrane morphogenesis.

Rabbit sarcoplasmic reticulum vesicles were fused into giant proteoliposomes in a medium of 0.1 M KCl, 10 mM Tris-maleate, pH 7.0, 10 micrograms ml-1 antipain, 10 micrograms ml-1 leupeptin, 25 IU per ml Trasylol, 3 mM NaN3, 3.75% PEG 1500 and 3% DMSO by brief exposure to 37 degrees C, followed by incubation for 4 h at 25 degrees C. Approximately 5-10% of the sarcoplasmic reticulum elements underwent fusion, forming single-walled spherical vesicles of 1-25 microns diameter, in which the polarity of the native membrane was preserved. The Ca(2+)-stimulated ATPase activity remained essentially unchanged after fusion. On exposure to decavanadate in a Ca(2+)-free medium the spherical vesicles assumed a corrugated appearance with the formation of long ridges separated by deep furrows that eventually pinched off longitudinally and separated into numerous long crystalline tubules of uniform (approximately 0.1 microns) diameter. The vanadate-induced transformation of giant vesicles into tubules implies that the geometry of the sarcoplasmic reticulum membrane is determined by the conformation of the Ca(2+)-ATPase.

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.

A tryptophan residue (W736) in the amino-terminus of the P-segment of domain II is involved in pore formation in Na(v)1.4 voltage-gated sodium channels.

Voltage-gated Na channels comprise four homologous domains each consisting of six transmembrane segments (S1-S6) linked by loops. The linkers between segments S5 and S6 in each domain (P-loops), denoted as SS1-SS2, form the pore of the channel. It is believed that the SS1 region of the P-loops dips into, while the SS2 region exits out of the membrane. We have reported previously that residues A728 and D730 (in SS1 of domain II) contribute to the external vestibule of the pore of the rat skeletal muscle Na channel (Na(v)1.4). In this study, we examined the role of a conserved neighbouring tryptophan residue at position 736 (W736) in the pore formation. The W736 residue of Na(v)1.4 was replaced by a cysteine using site-directed mutagenesis. Complementary RNAs encoding the wild-type and mutant channels were injected into Xenopus laevis oocytes and macroscopic Na(+) currents measured using the two-microelectrode voltage-clamp technique. The W736C mutant showed increased channel sensitivity to externally applied Cd(2+) and methanethiosulphonate-ethyltrimethylammonium (MTSET). Furthermore, micromolar concentrations of Cd(2+) reduced single-channel current amplitude in the Na(v)1.4/W736C mutant without affecting its voltage dependence. However, only small differences in tetrodotoxin and micro-conotoxin GIIIA affinity were observed between the wild-type and mutant channels. Replacing Na(+) with other cations - K(+), Li(+), Cs(+) or NH(4)(+) - did not change the ion permeation sequence of the Na(v)1.4/W736C mutant channel. The results suggest that W736 contributes to the formation of the pore, close to the mouth of the channel, but is not part of the selectivity filter.

Skeletal muscle regeneration after damage by needle penetration and trauma.

Skeletal muscles actually surround the dento-alveolar area. However, most dentists would be unaware that they damage skeletal muscle during routine procedures. Simple puncturing of buccinator muscle during an inferior alveolar block kills thousands of fibres. What happens to muscle fibres following such trauma? Pathology texts suggest that skeletal muscle does not regenerate and is replaced by fibrous scar tissue. However, for some decades it has been recognized that muscle fibres do in fact regenerate. In the early 1960s the "satellite" cell was discovered, lying between the muscle cell membrane and the external lamina. After 30 years of intensive research it has been clearly demonstrated that satellite cells are reserve mesenchyme cells which, once the adjacent muscle fibres are damaged, proliferate and provide a new population of young muscle cells, called "myoblasts". Myoblasts rapidly produce muscle specific proteins and fuse together in long chains, called "myotubes", which mature into typical muscle fibres.

The preparation of cell fusion-inducing proteoliposomes from purified glycoproteins of HVJ (Sendai virus) and chemically defined lipids.

Cell fusion-inducing (fusogenic) proteoliposomes of defined chemical composition were reconstituted from purified glycoproteins of hemagglutinating virus of Japan (Sendai virus) either with lipids extracted from the virus particles or with a chemically defined lipid mixture. Cell fusion reactions induced by the reconstituted system have several important characteristics similar to the virus-induced fusion reaction: fusogenic activity of the proteoliposomes depends on the presence of active fusion protein in the vesicles and, in the case of Ehrlich tumor cells, the fusion is almost completely inhibited by adding cytochalasin D to a final concentration of 4 microgram/ml. The only known difference between the original and reconstituted systems is that a greater amount of the latter is necessary for the same degree of fusogenic activity. Thus, the reconstituted system can be used as a model for the Sendai virus-induced fusion reaction. A lipid mixture (phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine:sphingomyelin = 1:2:1:1, by weight, and cholesterol equimolar to the total phospholipids) similar to that of the virion was active for reconstitution, whereas a mixture containing the same composition of phospholipids but no cholesterol, and ones containing cholesterol with only a single species of phospholipid were not reconstitutively active.

h1- and h2-calponins are not essential for norepinephrine- or sodium fluoride-induced contraction of rat aortic smooth muscle.

To investigate the controversial issue concerning the role of calponin in smooth muscle contraction, this study examined the relationship between smooth muscle calponin and the contraction of aortic rings from different strains of rats: Sprague-Dawley (SD), Wistar, and Wistar Kyoto (WKY). Western blot analysis demonstrated that h1- and h2-calponins are present in aortic smooth muscle from adult SD rats but not Wistar or WKY rats. Nevertheless, h1-calponin is detectable in stomach from Wistar rats, although at a much lower level compared with that in the SD rat stomach. This suggests that a repressed expression of the gene, instead of a simple null mutation, may have caused its absence from the aortic smooth muscle. Despite the presence or absence of calponin, the aortic smooth muscles from the different strains of rats all develop contractions in response to the physiological agonist norepinephrine (NE) and following activation with the plasma membrane receptor-independent NaF induction. The data indicate that h1- and h2-calponins are not essential for NE- and NaF-induced contractions in aortic smooth muscle. The calponin-positive adult SD rat aorta was found to be more sensitive in contractile response to NE and NaF inductions compared with the calponin-negative rat aortae. This may imply a potential modulator function of calponin in the contraction of smooth muscle, whereas other contractile protein isoform differences between these rat strains may also play a role.