Yves Maulet

Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Ca(v)1.1) and RyR(1), whereas in the heart the signal is carried from the cardiac-type DHPR (Ca(v)1.2) to RyR(2) by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Ca(v)1.2 or Ca(v)1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR(2). This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.

Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.

Calcium-dependent translocation of synaptotagmin to the plasma membrane in the dendrites of developing neurones

In neurones, the morphological complexity of the dendritic tree requires regulated growth and the appropriate targeting of membrane components. Accurate delivery of specific supplies depends on the translocation and fusion of transport vesicles. Vesicle SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and target membrane SNAREs play a central role in the correct execution of fusion events, and mediate interactions with molecules that endow the system with appropriate regulation. Synaptotagmins, a family of Ca(2+)-sensor proteins that includes neurone-specific members involved in regulating neurotransmitter exocytosis, are among the molecules that can tune the fusion mechanism. Using immunocytochemistry, confocal and electron microscopy, the localisation of synaptotagmin I in the dendrites of cultured rat hypothalamic neurones was demonstrated. Synaptotagmin labelling is concentrated at dendritic branch points, and in microprocesses. Following depolarisation, the N-terminal domain of synaptotagmin was detected at the extracellular surface of the dendritic plasma membrane. The insertion of synaptotagmin in the plasma membrane was elicited by L-type Ca(2+) channel activation and by mobilisation of the internal ryanodine-sensitive Ca(2+)stores. Furthermore, the localisation of L-type Ca(2+) channels and of ryanodine receptors, relative to the localisation of synaptotagmin in dendrites, suggests that both Ca(2+) entry and intracellular Ca(2+) stores may contribute to the fusion of dendritic transport vesicles with the membrane. Fusion is likely to involve SNAP-25 and syntaxin 1 as both proteins were also identified in dendrites. Taken together these results suggest a putative regulatory role of synaptotagmins in the membrane fusion events that contribute to shaping the dendritic tree during development.

Skeletal and cardiac ryanodine receptors bind to the Ca2+-sensor region of dihydropyridine receptor α1C subunit

In striated muscles, excitation–contraction coupling is mediated by the functional interplay between dihydropyridine receptor L‐type calcium channels (DHPR) and ryanodine receptor calcium‐release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross‐talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C‐terminal cytoplasmic domain of α1C. The interaction is restricted to the CB and IQ motifs involved in the calmodulin‐mediated Ca2+‐dependent inactivation of the DHPR, suggesting functional interactions between the two channels.

Evolution of Acetylcholinesterase Transcripts and Molecular Forms During Development in the Central Nervous System of the Quail

Abstract: We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes, and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiplemRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5‐kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is pre‐dominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65–70% in the neuroretina. This form is largely nonamhiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.

[21] Application of antisense techniques to characterize neuronal ion channels in vitro

Publisher Summary Current gene cloning and genomic initiatives provide neuroscientists with an exponentially increasing bank of genetic sequence data that details the molecular identity of novel brain proteins. Often in the initial absence of selective ligands, the functional properties of the more recently cloned brain proteins remain unclear. The antisense approach conceptually offers a solution to this problem. Multiple mechanisms have been proposed that describe the molecular events that precipitate gene-specific knockdown by antisense oligonucleotides. These include inhibition of transcription, translational arrest, disruption of ribonucleic acid (RNA) processing, and RNase H–mediated transcript degradation. Despite the many questions that remain unanswered, antisense oligonucleotides have been applied successfully in the central nervous system (CNS) research. This chapter discusses their in vitro applications and describes both the strategies and methods that have been developed and applied successfully to the studies of neuronal voltage-dependent ion channels. Wherever possible, the effects of antisense treatment have been examined at multiple levels—that is, transcription transduction, messenger RNA (mRNA), protein and/or functionally by electrophysiological measurements. Each level of analysis affords complementary data that when taken together greatly facilitate the final interpretation of results.