Calcium mediates dorsoventral patterning of mesoderm in Xenopus

AbstractCalcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes [1]. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction [2–5]. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that α1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity

Characterization of potassium channels in pancreatic β cells from ob/ob mice

AbstractThe patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic β cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal β cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent β cells. Thus, potassium channels in ob/ob pancreatic β cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability

DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses

Developmental regulation of the expression of sodium currents in Xenopus primary neurons

The electrophysiological properties of neurons are determined by the expression of defined complements of ion channels. Nonetheless, the regulation mechanisms of the expression of neuronal ion channels are poorly understood, due in part to the diversity of neuron subtypes. We explored the expression of voltage-gated currents of Xenopus primary spinal neurons unequivocally identified by means of single-cell RT-PCR. We found that identified spinal neurons exhibit heterogeneity in the temporal appearance of voltage-gated currents. Nevertheless, all neurons progress to similar functional phenotypes. A physiological feature is the onset and increase of the expression of sodium currents. To understand the mechanisms underlying this process, we studied the effect of a dominant negative form of the transcriptional silencer REST/NRSF and found that it associates to an increase in the density of sodium currents. This observation is compatible with a role of this factor in the regulation of gene expression in neurons. These experiments constitute a proof of principle for the feasibility of analyzing molecular mechanisms of the regulation of ion channel genes during early neuronal development and provide direct evidence of the role of REST/NRSF in the control of neuronal sodium channel expression

Modulation of the Kinetics of Inositol 1,4,5-Trisphosphate-Induced [Ca(2+)](i) Oscillations by Calcium Entry in Pituitary Gonadotrophs

Inositol 1,4,5-trisphosphate (InsP(3)) binds to its receptor channels and causes liberation of Ca(2+) from intracellular stores, frequently in an oscillatory manner. In addition to InsP(3), the activation and inactivation properties of these intracellular channels are controlled by Ca(2+). We studied the influence of Ca(2+) entry on the kinetics of InsP(3)-triggered oscillations in cytosolic calcium ([Ca(2+)](i)) in gonadotrophs stimulated with gonadotropin-releasing hormone, an agonist that activates InsP(3) production. The natural expression of voltage-gated Ca(2+) channels (VGCC) in these cells was employed to manipulate Ca(2+) entry by voltage clamping the cells at different membrane potentials (V(m)). Under physiological conditions, the frequency of the GnRH-induced oscillations increased with time, while the amplitude decreased, until both reached stable values. However, in cells with V(m) held at -50 mV or lower, both parameters progressively decreased until the signal was abolished. These effects were reverted by a depolarization of the membrane positive to -45 mV in both agonist- and InsP(3)-stimulated gonadotrophs. Depolarization also led to an increase in the fraction of time during which the [Ca(2+)](i) remained elevated; this effect originated from both an increase in the mean duration of spikes and a decrease in the interval between spikes. The frequency and amplitude of spiking depended on the activity of VGCC, but displayed different temporal courses and voltage relationships. The depolarization-driven recovery of the frequency was instantaneous, whereas the recovery of the amplitude of spiking was more gradual. The midpoints of the V(m) sensitivity curve for amplitude and duration of spiking (-15 mV) were close to the value observed for L-type Ca(2+) current and for depolarization-induced increase in [Ca(2+)](i), whereas this parameter was much lower (-35 mV) for interval between spikes and frequency of oscillations. These observations are compatible with at least two distinct effects of Ca(2+) entry on the sustained [Ca(2+)](i) oscillations. Calcium influx facilitates its liberation from intracellular stores by a direct and instantaneous action on the release mechanism. It also magnifies the Ca(2+) signal and decreases the frequency because of its gradual effect on the reloading of intracellular stores