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Ryanodine treatment - a plant alkaloid known to be a powerful inhibitor of muscle contraction - changes the ionic selectivity and conductance of several types of K+ channels in muscle cells. Available data provide evidence that the effects of ryanodine on K+ channel properties are not secondary to Ca2+ release from sarcoplasmic reticulum, but result from a direct interaction of the alkaloid with the channel protein. In the present investigations we applied the whole-cell patch-clamp technique to study ryanodine effects on the properties of Kv1.3 channels, which are present in lymphocytes and rat brain cells. The effects of ryanodine applied in the concentration range 10-5-10-4 M on the ionic selectivity, conductance, gating and kinetics of Kv1.3 channels expressed in human T lymphocytes were examined. Our data provide evidence that none of these properties was changed upon ryanodine treatment. Altogether, our data support the notion that Ryanodine may interact with various types of K+ channels differently. The different response to Ryanodine treatment might be another pharmacological feature delineating differences among various types of K+ channels.
The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 μM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 μM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 μM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82±13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11±3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 μM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 μM EGTA, 10 μM ryanodine or extracellular application of 10 μM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.
The latest developed classes of synthetic insecticides – phthalic acid diamides and antranilic diamides, acting as ryanodine receptor modulators, are discussed. The mode of their activity, the insecticidal properties and characteristics of their representatives is reviewed.
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