Continual remodeling of postsynaptic density and its regulation by synaptic activity.

A postsynaptic density (PSD) protein, PSD-95, was tagged with green fluorescent protein (GFP-PSD-95) and expressed in cultured hippocampal neurons using recombinant adenoviruses. GFP-PSD-95 was selectively localized to excitatory postsynaptic sites. Time-lapse fluorescence imaging of hippocampal neurons revealed that >20% of GFP-PSD-95 clusters turned over within 24 hours. The appearance rate of clusters was higher than the disappearance rate, and this difference accounted for the gradual increase of the cluster density observed in culture. Dynamics of PSD-95 clusters were also inhibited by blockers of excitatory synaptic transmission. Continual PSD turnover and its regulation by synaptic activity may be important in activity-dependent remodeling of neuronal connections.

Possible mediation of G-proteins in cold-sensory transduction in

The possible involvement of G-proteins in cold-sensory transduction was examined using voltage-clamped Paramecium multimicronucleatum into which non-hydrolyzable guanosine nucleotide analogues had been applied intracellularly. Guanosine-5'-O-3-thiotriphosphate, guanosine-5'-O-2-thiodiphosphate and aluminium fluoride all reduced the transient inward current in response to cooling, suggesting the possibility that G-proteins mediate cold-sensory transduction. Internal application of a Ca2+ chelator, EGTA, also reduced the current response. In addition to their effect on reducing the cold-sensory response, application of these chemicals modulated both the resting potential and the membrane conductance. Possible correlations between G-protein activity and the regulation of intracellular Ca2+ levels are discussed.

Cell models of Blepharisma: Ca(2+)-dependent modification of ciliary movement and cell elongation.

Ionic mechanisms were examined with reference to modification of swimming velocity and cell elongation in Triton-extracted cell models of Blepharisma. The extracted cells swam forward at Ca(2+) concentrations below 10(-6) M. The forward swimming velocity of the cell models increased with a decreased Ca(2+) concentration in the surrounding medium. At Ca(2+) concentrations above 10(-6) M, the models swam backward or rotated. The elongation of the models occurred at Ca(2+) concentrations below 10(-7) M. Results suggest that swimming velocity, cell elongation and contraction of intact cells may be regulated by intracellular Ca(2+) concentration.

Cold-sensitive Ca2+ influx in Paramecium.

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.

Cold-induced decrease of K+ conductance and its inhibition by a calmodulin antagonist, W-7, in Paramecium tetraurelia.

Under voltage clamp, Paramecium tetraurelia was used to examine the cold-induced inward current and its inhibition by a calmodulin antagonist, W-7 [N-(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide]. Cooling of the cell caused an inward current. The amplitude of the current was increased as the membrane potential was made more positive than the resting potential, and it was significantly blocked by using CsCl-filled electrodes and tetraethylammonium in the bath solution, suggesting that the current was accompanied mainly by a decrease in K+ conductance. The cold-induced inward current was reversibly inhibited by the external application of W-7 in a concentration-dependent manner. EGTA-microinjection into the cell also reduced the current. These results indicate that the decrease in K+ conductance induced by cooling is Ca(2+)-dependent and is inhibited by W-7.

Defect of cold-sensitive response in calmodulin mutants of Paramecium and the restoration by cyclic nucleotide.

Wild type and calmodulin mutants (cam) of Paramecium tetraurelia were examined for cold-sensitive responses. Among mutants tested, cam12 and cam13 mutants, which have substitutions in N-terminal lobe of calmodulin molecule, reduced both responses in the swimming and the membrane potential. Under voltage clamp conditions, the cooling stimulus to the wild type cell induced a transient inward current whose amplitude increased with the rate of temperature drop. The cam12 cell did not induce inward currents in response to cooling with a rate slower than -0.4 degree C/s. The reduced current response of cam12 mutant was restored by an external application of a phosphodiesterase inhibitor, theophylline. Also, an intracellular injection of hydrolysis-resistant cyclic nucleotides, either 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5'-cyclic monophosphate (8-Br-cGMP), restored the current response. Such restoration was accompanied by shifts of the resting potential to hyperpolarized levels and by an increase in the membrane conductance. The results suggest the possibility that calmodulin and cyclic nucleotide regulate K+ channels responsive to the cooling stimulus.