Physical Therapy Interventions of an Electrical Burn Injury-Afflicted Patient: A Case Report.

Electrical injuries are uncommon but not completely rare. It is most prevalent in the male population, although females are also affected in the workplace or household-related activities. These injuries usually occur in situations where proper precautions are not taken by the individual and also appropriate safety drills and education for personnel are not carried out. Electrical burns affecting children are very rare, but when they do occur, it is usually due to accidental contact with exposed electrical sources. In this patient, there were severe levels of secondary complications following the burn injury. The patient developed blood infections and also was hampered in doing a variety of activities of daily living. The patient was diagnosed with 45%-50% body surface area (BSA) covered with burns, which suggests its severe nature. Treatment focuses on preventing wound infection, managing the excruciating amount of pain, preventing complications of immobility, promoting mobility as much as the patient can, and also educating the patient and the family members.

Mechanism of modulation of AMPA receptors by TARP-γ8.

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

The structural arrangement at intersubunit interfaces in homomeric kainate receptors.

Kainate receptors are glutamate-gated cation-selective channels involved in excitatory synaptic signaling and are known to be modulated by ions. Prior functional and structural studies suggest that the dimer interface at the agonist-binding domain plays a key role in activation, desensitization, and ion modulation in kainate receptors. Here we have used fluorescence-based methods to investigate the changes and conformational heterogeneity at these interfaces associated with the resting, antagonist-bound, active, desensitized, and ion-modulated states of the receptor. These studies show that in the presence of Na+ ions the interfaces exist primarily in the coupled state in the apo, antagonist-bound and activated (open channel) states. Under desensitizing conditions, the largely decoupled dimer interface at the agonist-binding domain as seen in the cryo-EM structure is one of the states observed. However, in addition to this state there are several additional states with lower levels of decoupling. Replacing Na+ with Cs+ does not alter the FRET efficiencies of the states significantly, but shifts the population to the more decoupled states in both resting and desensitized states, which can be correlated with the lower activation seen in the presence of Cs+.

The structure-energy landscape of NMDA receptor gating.

N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.

Stargazin Modulation of AMPA Receptors.

Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of the receptor and how this distribution is affected by stargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor.

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Phospholamban and sarcolipin: Are they functionally redundant or distinct regulators of the Sarco(Endo)Plasmic Reticulum Calcium ATPase?

In muscle, the Sarco(Endo)plasmic Reticulum Calcium ATPase (SERCA) activity is regulated by two distinct proteins, PLB and SLN, which are highly conserved throughout vertebrate evolution. PLB is predominantly expressed in the cardiac muscle, while SLN is abundant in skeletal muscle. SLN is also found in the cardiac atria and to a lesser extent in the ventricle. PLB regulation of SERCA is central to cardiac function, both at rest and during extreme physiological demand. Compared to PLB, the physiological relevance of SLN remained a mystery until recently and some even thought it was redundant in function. Studies on SLN suggest that it is an uncoupler of the SERCA pump activity and can increase ATP hydrolysis resulting in heat production. Using genetically engineered mouse models for SLN and PLB, we showed that SLN, not PLB, is required for muscle-based thermogenesis. However, the mechanism of how SLN binding to SERCA results in uncoupling SERCA Ca(2+) transport from its ATPase activity remains unclear. In this review, we discuss recent advances in understanding how PLB and SLN differ in their interaction with SERCA. We will also explore whether structural differences in the cytosolic domain of PLB and SLN are the basis for their unique function and physiological roles in cardiac and skeletal muscle.

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport.

The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca(2+) transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues (2)ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.

Sarcolipin overexpression improves muscle energetics and reduces fatigue.

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

Sarcolipin Is a Key Determinant of the Basal Metabolic Rate, and Its Overexpression Enhances Energy Expenditure and Resistance against Diet-induced Obesity.

Sarcolipin (SLN) is a novel regulator of sarcoplasmic reticulum Ca(2+) ATPase (SERCA) in muscle. SLN binding to SERCA uncouples Ca(2+) transport from ATP hydrolysis. By this mechanism, SLN promotes the futile cycling of SERCA, contributing to muscle heat production. We recently showed that SLN plays an important role in cold- and diet-induced thermogenesis. However, the detailed mechanism of how SLN regulates muscle metabolism remains unclear. In this study, we used both SLN knockout (Sln(-/-)) and skeletal muscle-specific SLN overexpression (Sln(OE)) mice to explore energy metabolism by pair feeding (fixed calories) and high-fat diet feeding (ad libitum). Our results show that, upon pair feeding, Sln(OE) mice lost weight compared with the WT, but Sln(-/-) mice gained weight. Interestingly, when fed with a high-fat diet, Sln(OE) mice consumed more calories but gained less weight and maintained a normal metabolic profile in comparison with WT and Sln(-/-) mice. We found that oxygen consumption and fatty acid oxidation were increased markedly in Sln(OE) mice. There was also an increase in both mitochondrial number and size in Sln(OE) muscle, together with increased expression of peroxisome proliferator-activated receptor δ (PPARδ) and PPAR γ coactivator 1 α (PGC1α), key transcriptional activators of mitochondrial biogenesis and enzymes involved in oxidative metabolism. These results, taken together, establish an important role for SLN in muscle metabolism and energy expenditure. On the basis of these data we propose that SLN is a novel target for enhancing whole-body energy expenditure.

Sarcolipin protein interaction with sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is distinct from phospholamban protein, and only sarcolipin can promote uncoupling of the SERCA pump.

Sarco(endo)plasmic reticulum Ca(2+)ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547-554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575-1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the Vmax of SERCA-mediated Ca(2+) uptake but not the pump affinity for Ca(2+); 2) SLN can bind to SERCA in the presence of high Ca(2+), but PLB can only interact to the ATP-bound Ca(2+)-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca(2+) causes uncoupling of the SERCA pump and increased heat production.

Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals.

The role of skeletal muscle in nonshivering thermogenesis (NST) is not well understood. Here we show that sarcolipin (Sln), a newly identified regulator of the sarco/endoplasmic reticulum Ca(2+)-ATPase (Serca) pump, is necessary for muscle-based thermogenesis. When challenged to acute cold (4 °C), Sln(-/-) mice were not able to maintain their core body temperature (37 °C) and developed hypothermia. Surgical ablation of brown adipose tissue and functional knockdown of Ucp1 allowed us to highlight the role of muscle in NST. Overexpression of Sln in the Sln-null background fully restored muscle-based thermogenesis, suggesting that Sln is the basis for Serca-mediated heat production. We show that ryanodine receptor 1 (Ryr1)-mediated Ca(2+) leak is an important mechanism for Serca-activated heat generation. Here we present data to suggest that Sln can continue to interact with Serca in the presence of Ca(2+), which can promote uncoupling of the Serca pump and cause futile cycling. We further show that loss of Sln predisposes mice to diet-induced obesity, which suggests that Sln-mediated NST is recruited during metabolic overload. These data collectively suggest that SLN is an important mediator of muscle thermogenesis and whole-body energy metabolism.

Invasive capabilities of Campylobacter jejuni strains isolated in Bahrain: molecular and phenotypic characterization.

The association between putative virulence genes in Campylobacter jejuni clinical isolates, in vitro invasive capability and severity of infection is yet to be clearly described. We have characterized three virulence genes and correlated their presence with the severity of infection and in vitro invasiveness. We studied eight C. jejuni strains isolated from patients whose clinical data were scored to determine severity of infection. Cytolethal distending toxin (cdtB), invasion associated marker (iam) and Campylobacter invasion antigen (ciaB) genes were detected by PCR and INT407 cells used for invasion assays. Two strains positive for all three genes were the most invasive and isolated from patients with the most severe infection. Four strains positive for two genes and two strains negative for all the three genes were identified. The two cdtB(+ve)/ciaB(+ve) strains were more invasive than the cdtB(+ve)/iam(+ve) strains. One of the cdtB(-ve)/ciaB(-ve) strains showed invasion levels similar to cdtB(+ve)/ciaB(+ve) strains, but the second strain had a non-invasive phenotype. The findings indicate a correlation between in vitro invasive capability, and the presence of all three genes. The pattern of association between invasiveness and molecular characterization suggests that the ciaB gene confers a more invasive capability.