Caroline Strube

α1H mRNA in single skeletal muscle fibres accounts for T‐type calcium current transient expression during fetal development in mice

Calcium channels are essential for excitation‐contraction coupling and muscle development. At the end of fetal life, two types of Ca2+ currents can be recorded in muscle cells. Whereas L‐type Ca2+ channels have been extensively studied, T‐type channels have been poorly characterized in skeletal muscle. We describe here the functional and molecular properties of T‐type calcium channels in developing mouse skeletal muscle. The T‐type current density increased transiently during prenatal myogenesis with a maximum at embryonic day E16 followed by a drastic decrease until birth. This current showed similar electrophysiological and pharmacological properties at all examined stages. It displayed a wide window current centred at about −35 and −55 mV in 10 and 2 mm external Ca2+, respectively. Activation and inactivation kinetics were fast (3 and 16 ms, respectively). The current was inhibited by nickel and amiloride with an IC50 of 5.4 and 156 μm, respectively, values similar to those described for cloned T‐type α1H channels. Whole muscle tissue RT‐PCR analysis revealed mRNAs corresponding to α1H and α1G subunits in the fetus but not in the adult. However, single‐fibre RT‐PCR demonstrated that only α1H mRNA was present in prenatal fibres, suggesting that the α1G transcript present in muscle tissue must be expressed by non‐skeletal muscle cells. Altogether, these results demonstrate that the α1H subunit generates functional T‐type calcium channels in developing skeletal muscle fibres and suggest that these channels are involved in the early stages of muscle differentiation.

Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells.

Caveolae and transverse (T‐) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T‐system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation–contraction (E–C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T‐tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl‐β‐cyclodextrin (MβCD), a cholesterol‐sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1–3 mm MβCD for 1 h at 37°C led to a significant reduction in caveolae and T‐tubule areas and to a decrease in cell membrane electrical capacitance. In whole‐cell voltage‐clamp experiments, the L‐type Ca2+ current amplitude was significantly reduced, and its voltage dependence was shifted ∼15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca2+ transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E–C coupling. In contrast, T‐type Ca2+ current was not affected by MβCD treatment. Most of the L‐type Ca2+ conductance reduction and E–C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T‐tubules. However, the effects on L‐type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L‐type current without any change in the voltage dependence of charge movement and Ca2+ transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Ozone inhalation activates stress-responsive regions of the CNS.

J. Neurochem. (2011) 117, 961–972.

Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics.

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.

Early expression of AMPA receptors and lack of NMDA receptors in developing rat climbing fibre synapses

Whether nascent glutamatergic synapses acquire their AMPA receptors constitutively or via a regulated pathway triggered by pre‐existing NMDA receptor activation is still an open issue. Here, we provide evidence that some glutamatergic synapses develop without expressing NMDA receptors. Using immunocytochemistry, we showed that synapses between developing rat climbing fibres and Purkinje cells expressed GluR2‐containing AMPA receptors as soon as they were formed (i.e. on embryonic day 19) but never carried detectable NMDA receptors. This was confirmed by electrophysiological recordings. Excitatory synaptic currents were recorded in Purkinje cells as early as P0. However, no NMDA receptor‐mediated component was found in either spontaneous or evoked synaptic responses. In addition, we ruled out a possible role of extrasynaptic NMDA receptors by showing that AMPA receptor clustering at nascent climbing fibre synapses was not modified by chronic in utero NMDA receptor blockade.

Functional Expression of the L-Type Calcium Channel in Mice Skeletal Muscle during Prenatal Myogenesis

The densities of skeletal muscle intramembrane charge movement and macroscopic L-type Ca(2+) current have been shown to increase during prenatal development. In the present work, the electrophysiological characteristics of L-type Ca(2+) channels were analyzed over the embryonic period E14 to E19 using the whole-cell and cell-attached procedures. At the macroscopic level, the whole-cell L-type Ca(2+) conductance increased 100% between E14 and E19. This enhancement was accompanied by a small negative shift of the voltage dependence and a marked acceleration of the inactivation kinetics. At the single-channel level, the unitary conductance decreased significantly from 13.2 +/- 0.1 pS (n = 8) at E14 to 10.7 +/- 0.3 pS (n = 7) at E18 and the open probability was multiplied by 2. No significant change of the density of functional channels was observed during the same period. In contrast to the density of intramembrane charge movement, which, under the same conditions, has been shown to increase between 16 and 19 days, L-type Ca(2+) channels properties change mostly between 14 and 16 days. Taken together, these results suggest that the two functions of the dihydropyridine receptor are carried by two different proteins which could be differentially regulated by subunit composition and/or degree of phosphorylation.

Intramembrane charge movement in developing skeletal muscle cells from fetal mice

The development of intramembrane charge movement was studied in freshly isolated skeletal muscle cells from 13- to 19-day-old mouse fetuses. Charge movement was present in myotubes from 13-day-old fetuses. The relationship between charge movement and membrane potential could be described by a two-state Boltzmann equation. The amount of maximum charge movement (Qmax) increased substantially with the age of the fetuses from 2.84±0.39 nC/μF (n=10) at day 13 to 10.01±0.97 nC/μF (n=15) at day 19. Nifedipine (1 μM) consistently reduced Qmax by 33±2% (n=37) of the control value at each age studied. Increasing the concentration of nifedipine to 20 μM had no further effect, suggesting that the charge movement in developing myotubes consists of at least two components: a nifedipine-sensitive charge movement (Qns) and a nifedipine-resistant one (Qnr). Both Qns and Qnr increased exponentially with a distinct enhancement of rate at day 16.

Reduced intramembrane charge movement in the dysgenic skeletal muscle cell.

Intramembrane charge movement in skeletal muscle cells has been proposed to underlie the process leading to Ca release from the sarcoplasmic reticulum. A number of recent studies suggest that the dihydropyridine receptor located in the transverse-tubular membrane is responsible for the generation of intramembrane charge movement. The skeletal muscle cell of the mutant mouse with “Muscular Dysgenesis” is characterized by absence of excitation-contraction coupling. Here we investigated the charge movement in freshly dissociated skeletal muscle cells from dysgenic mice. In 9 out of 34 dysgenic mouse cells the charge movement was completely absent, in the remaining cells the charge movement was never more than 30 % of control. The amount of maximum charge movement (Qmax) in mutant muscle cells was less than 30 % of Qmax in normal muscle. Nifedipine, a dihydropyridine derivative, reduced the amount of charge movement in normal muscle cells but it was less effective on charge movement in mutant muscle cells. We conclude that there is an alteration of nifedipine-sensitive charge movement in the skeletal muscle cells from the mutant mice.

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca2+]i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca2+ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca2+]i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 μM lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca2+]i by stimulating Ca2+ release from the sarcoplasmic reticulum independently of extracellular Ca2+ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca2+ entry through calcium channels was suppressed.

Effect of SR33557 on Intramembrane Charge Movement in Normal and‘Muscular Dysgenesis’Mouse Skeletal Muscle Cells

It has been reported that the indolizinsulphone SR33557, which binds to a site on the α1 subunit of the dihydropyridine receptor, blocks both L‐type calcium channel activity and contraction in skeletal muscle. Moreover, we know that charge movement plays a key role in the mechanism of excitation‐contraction coupling and in controlling the opening of L‐type calcium channels. We demonstrate here that SR33557 reduces intramembrane charge movement in skeletal muscle from normal mice with an IC50 of ‐10 nM. The drug does not completely inhibit charge movement since ‐20% of total charge movement persists even in the presence of 30 μM SR33557. However, the SR33557‐sensitive charge component is more important than the dihydropyridine‐sensitive one. Surprisingly, SR33557 also reduces intramembrane charge movement in dysgenic myotubes which are characterized by a very strong reduction of the number of dihydropyridine binding sites. In these muscles, 10 μM SR33557 reduces ‐40% of total charge movement. These observations suggest the presence of a new component of charge movement which is sensitive to SR33557 but insensitive to nifedipine. This component is also present in dysgenic myotubes, and it could be produced by the lower molecular weight α1, subunit described by Malouf, N. N., McMahon, D. K., Hainsworth, C. N. and Kay, B. K. (1992) (Neuron, 8, 899–906).