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1.
Biophys J ; 99(2): 407-16, 2010 Jul 21.
Article in English | MEDLINE | ID: mdl-20643058

ABSTRACT

To clarify the molecular mechanisms behind quantal Ca2+ release, the graded Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptor (InsP3R) channels responding to incremental ligand stimulation, single-channel patch-clamp electrophysiology was used to continuously monitor the number and open probability of InsP3R channels in the same excised cytoplasmic-side-out nuclear membrane patches exposed alternately to optimal and suboptimal cytoplasmic ligand conditions. Progressively more channels were activated by more favorable conditions in patches from insect cells with only one InsP3R gene or from cells solely expressing one recombinant InsP3R isoform, demonstrating that channels with identical primary sequence have different ligand recruitment thresholds. Such heterogeneity was largely abrogated, in a fully reversible manner, by treatment of the channels with sulfhydryl reducing agents, suggesting that it was mostly regulated by different levels of posttranslational redox modifications of the channels. In contrast, sulfhydryl reduction had limited effects on channel open probability. Thus, sulfhydryl redox modification can regulate various aspects of intracellular Ca2+ signaling, including quantal Ca2+ release, by tuning ligand sensitivities of InsP3R channels. No intrinsic termination of channel activity with a timescale comparable to that for quantal Ca2+ release was observed under any steady ligand conditions, indicating that this process is unlikely to contribute.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Animals , Calcium Signaling/drug effects , Cell Line , Chickens , Dithiothreitol/pharmacology , Insecta/cytology , Ion Channel Gating/drug effects , Ligands , Models, Biological , Oxidation-Reduction/drug effects , Rats , Recombinant Proteins/metabolism , Sulfhydryl Compounds/metabolism
2.
Int J Dev Biol ; 53(1): 101-8, 2009.
Article in English | MEDLINE | ID: mdl-19123131

ABSTRACT

Thyroid hormones--particularly triiodothyronine, T3--play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre- or pro-metamorphic stages. Exposure to T3 in the water induced a rapid (beginning within 5 min) and significant decrease (approximately 20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI<2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI> or =2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.


Subject(s)
Locomotion/drug effects , Locomotion/physiology , Metamorphosis, Biological/drug effects , Rana catesbeiana/growth & development , Swimming/physiology , Triiodothyronine/pharmacology , Animals , Cheek/physiology , Electrophysiological Phenomena
3.
EMBO Rep ; 8(11): 1044-51, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17932510

ABSTRACT

The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel is engaged by thousands of plasma membrane receptors to generate Ca(2+) signals in all cells. Understanding how complex Ca(2+) signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP(3) and Ca(2+), which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP(3)R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca(2+) regulation and unexpected ligand cooperativity. The kinetics of Ca(2+)-mediated channel inhibition showed the single-channel bases for fundamental Ca(2+) release events and Ca(2+) release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca(2+) signals.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Ion Channel Gating/physiology , Animals , Cell Line , Insecta , Ligands , Patch-Clamp Techniques
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