The noradrenaline (NA)‐activated response was investigated in neurones acutely dissociated from the rat locus coeruleus (LC) using nystatin‐perforated, conventional whole‐cell and inside‐out patch recording modes under current‐ and voltage‐clamp conditions. Under current‐clamp conditions, NA hyperpolarized the LC neurones, abolishing the spontaneous action potentials. In voltage‐clamp studies, NA induced an inwardly rectifying K+ current (INA) in a concentration‐dependent manner with a half‐maximum effective concentration of 2.2 × 10−7 M. I NA was mimicked by the α2‐agonist UK14304 but was inhibited by either the α2B/α2C antagonist ARC239 or the α1‐ and α2B/α2C antagonist prazosin, suggesting the contribution of α2B/α2C adrenoceptors. I NA was inhibited by the intracellular application of GDPβS but fully activated by intracellular perfusion of GTPγS. In the inside‐out recording mode, the application of GTP to the cytoplasmic side of the patch membrane markedly enhanced the open probability of the NA‐activated single channels which represented the inwardly rectifying properties. These results indicate that the activation of α2B/α2C adrenoceptors coupled with GTP‐binding protein directly activates the inwardly rectifying K+ currents in rat LC neurones, thus resulting in a decrease in the spontaneous firing activities.
The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.T he hexactinellids are a circumglobal group of predominantly deep sea sponges. Exhibiting a diverse array of morphologies, the skeletal systems of hexactinellids, which are composed of amorphous hydrated silica (the other silica-forming sponge class is the Demospongiae), range in structural complexity from loose aggregations of individual skeletal elements (spicules) to complex, hierarchically ordered lattices. Hexactinellids have evolved the ability to colonize either rocky substrates or soft sediments and exhibit remarkable skeletal modifications to accomplish these feats (1). For example, the sediment-dwelling hexactinellids produce bundles of long fibrillar anchor spicules that form robust holdfast structures. These anchor spicules exhibit surprising flexibility and damage tolerance and have served as useful model systems for investigating high performance silica-based organic-inorganic biological composites (2-4). In one representative genus, Euplectella (Fig. 1), the constituent spicules are assembled into a highly regular cylindrical lattice that exhibits multiple levels of structural hierarchy spanning from the nanoscale to the macroscale. The lattice is composed of two interpenetrating grid-like networks of no...
Spontaneous miniature outward currents (SMOCs) were observed in mechanically dissociated rat Meynert neurons using nystatin perforated patch recordings under voltage‐clamp conditions. SMOCs were blocked by apamin, a selective blocker of small conductance Ca2+‐activated K+ (SK) channels, but not by blockers for other types of Ca2+‐activated K+ channel. Ryanodine (10‐100 μm) reduced both the amplitude and frequency of SMOCs. Caffeine (1 mm) increased the SMOC frequency. Blockers of the sarco/endoplasmic reticulum Ca2+‐ATPase completely abolished SMOCs, indicating a requirement for functioning sarco/endoplasmic reticulum (SR/ER) Ca2+ stores. Both Cd2+‐containing and Ca2+‐free solutions partially inhibited SMOC frequency, a result which suggests that Ca2+ influx contributes to, but is not essential for, SMOC generation. Thus, SMOCs are SK currents linked to ryanodine‐ and caffeine‐sensitive SR/ER Ca2+ stores, and are only indirectly influenced by extracellular Ca2+ influx. The development of this new, minimally invasive mechanical dissociation method has revealed that SMOCs are common in native CNS neurons.
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