A1152D mutation of the Na+ channel causes paramyotonia congenita and emphasizes the role of DIII/S4–S5 linker in fast inactivation

Missense mutations in the human skeletal muscle Na⁺ channel α subunit (hSkM1) are responsible for a number of muscle excitability disorders. Among them, paramyotonia congenita (PC) is characterized by episodes of muscle stiffness induced by cold and aggravated by exercise. We have identified a new PC-associated mutation, which substitutes aspartic acid for a conserved alanine in the S4–S5 linker of domain III (A1152D). This residue is of particular interest since its homologue in the rat brain type II Na⁺ channel has been suggested as an essential receptor site for the fast inactivation particle. To identify the biophysical changes induced by the A1152D mutation, we stably expressed hSkM1 mutant or wild-type (WT) channels in HEK293 (human embryonic kidney) cells, and recorded whole-cell Na⁺ currents with the patch-clamp technique. Experiments were performed both at 21 and 11°C to better understand the sensitivity to cold of paramyotonia. The A1152D mutation disrupted channel fast inactivation. In comparison to the WT, mutant channels inactivated with slower kinetics and displayed a 5 mV depolarizing shift in the voltage dependence of the steady-state. The other noticeable defect of A1152D mutant channels was an accelerated rate of deactivation from the inactivated state. Decreasing temperature by 10°C amplified the differences in channel gating kinetics between mutant and WT, and unveiled differences in both the sustained current and channel deactivation from the open state. Overall, cold-exacerbated mutant defects may result in a sufficient excess of Na⁺ influx to produce repetitive firing and myotonia. In the light of previous reports, our data point to functional as well as phenotypic differences between mutations of conserved S4–S5 residues in domains II and III of the human skeletal muscle Na⁺ channel.     

Three potassium currents in mouse motor nerve terminals

A study of the K conductance of the presynaptic membrane has been performed in thetriangularis sterni muscle of the mouse. External currents generated in the presynaptic terminals upon invasion by action potentials have been recorded using microelectrodes inserted into the perineurium of preterminal nerve bundles. The voltage-dependent K current could be pharmacologically dissected into fast (IK_f) and slow (IK_s) components. While both are sensitive to 3,4-diaminopyridine (3,4-DAP), only IK_f is sensitive to tetraethylammonium (TEA). Uranyl (100–200 M) and guanidine (5–10 mM) produced a near complete block of IK_f and IK_s, which can explain their facilitatory effect upon transmitter release. The third K current of presynaptic terminals is Ca²⁺-dependent, but was activated also by Sr²⁺. This current could be suppressed by nanomolar doses of charybdotoxin; it is also sensitive to TEA but not to 3,4-DAP, uranyl or guanidine.     

Cold extends electromyography distinction between ion channel mutations causing myotonia

OBJECTIVE: Myotonias are inherited disorders of the skeletal muscle excitability. Nondystrophic forms are caused by mutations in genes coding for the muscle chloride or sodium channel. Myotonia is either relieved or worsened by repeated exercise and can merge into flaccid weakness during exposure to cold, according to causal mutations. We designed an easy electromyography (EMG) protocol combining repeated short exercise and cold as provocative tests to discriminate groups of mutations. METHODS: Surface-recorded compound muscle action potential was used to monitor muscle electrical activity. The protocol was applied on 31 unaffected control subjects and on a large population of 54 patients with chloride or sodium channel mutations known to cause the different forms of myotonia. RESULTS: In patients, repeated short exercise test at room temperature disclosed three distinct abnormal patterns of compound muscle action potential changes (I-III), which matched the clinical symptoms. Combining repeated exercise with cold exposure clarified the EMG patterns in a way that enabled a clear correlation between the electrophysiological and genetic defects. INTERPRETATION: We hypothesize that segregation of mutations into the different EMG patterns depended on the underlying pathophysiological mechanisms. Results allow us to suggest EMG guidelines for the molecular diagnosis, which can be used in clinical practice.     

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.     

Terminal sprouting in mouse neuromuscular junctions poisoned with botulinum type A toxin: morphological and electrophysiological features.

Functional properties of terminal sprouts elicited by an in vivo injection of Clostridium botulinum type A toxin were studied in endplates of the Levator auris longus muscle of the mouse poisoned from a few days to 28 days beforehand. For this purpose, morphological observations of the extent of terminal sprouts and localization of acetylcholine receptors was performed in whole mount preparations. Sprouts appeared as thin unmyelinated filaments that run usually parallel to the longitudinal axis of the muscle fibres; labelling acetylcholine receptors revealed their line-shaped accumulation co-localized with the sprouts. In addition, presynaptic membrane currents elicited by nerve stimulation were recorded by external electrodes applied under visual control onto the membrane of pre-existing motor endings and newly formed sprouts. These recordings showed the presence of widespread triphasic waveforms which indicated active impulse propagation of the action potential over most of the length of the poisoned endings. Ca2+ influx and Ca2(+)-dependent K+ currents in the sprout membrane were found to be similar to those described in unpoisoned endings. The presence of normal Ca2+ influx, upon active depolarization, in the terminal sprout membranes together with the localization of acetylcholine receptors in front of these membranes, indicates that the terminal sprouts may play a role in the recovery of neuromuscular transmission after Clostridium botulinum poisoning.     

Antidiabetic activity of aqueous leaf extract of Atriplex halimus L. (Chenopodiaceae) in streptozotocin-induced diabetic rats

Objective

To investigate the antidiabetic effect of A. halimus leaf in streptozotocin-induced diabetic rats.

Methods

The aqueous extract of the plant leaf was tested for its efficacy in streptozotocin-induced diabetic rats. The extract was evaluated for its acute and short term general toxicity in male mice and for its antihyperglycemic activity using glucose tolerance test in rats. The aqueous extract was subjected to phytochemical screening and determination of total phenolic contents.

Results

The statistical data indicated the significant increase in the body weight and decrease in the blood glucose and hepatic levels. The total protein level was significantly increased when treated with the extract.

Conclusions

These results suggest that the aqueous leaf extract of A. halimus has beneficial effects in reducing the elevated blood glucose level and hepatic levels in streptozotocin-induced diabetic rats.     

Invasive central nervous system aspergillosis in bone marrow transplantation recipients: an overview

Invasive central nervous system aspergillosis is being seen with an increased frequency, particularly due to the increased number of immunosuppressed patients. The major cause of invasive central nervous system aspergillosis is bone marrow transplantation. In most cases, aspergillosis develops in the paranasal sinuses and in the lungs, and secondarily spreads to the brain. Imaging of cerebral aspergillosis may present different patterns depending on the lesion's age and the immunologic status of the patient. Lesions of the spinal cord are far less common but has been encountered in our series. In this article we review the clinical and radiologic features of aspergillosis affecting the central nervous system in patients who underwent bone marrow transplantation. Different CT and MR patterns are presented, including pertinent clinical and pathologic material. Significant morbidity and mortality can be associated with this fungal infection, and it is therefore incumbent upon the radiologist to identify intracranial aspergillosis as early as possible so that appropriate therapy can be administered. Electronic Publication