University of Heidelberg (2006) Proc Physiol Soc 4, PC3
Poster Communications: Claude Collet1, Luc Belzunces1
1. UMR 406 Ecologie des Invertébrés, Institut National de la Recherche Agronomique, Avignon, France.
Electrical properties of adult honeybee skeletal muscle fibres enzymatically isolated from the metathoracic leg tibia were explored with the whole-cell patch-clamp technique. In a physiological-like extracellular solution, pressure perfusion (2 psi) with L-glutamate, an excitatory neurotransmitter at the neuromuscular synapse of invertebrates, induced fast activation of an inward current in fibres voltage clamped at a resting membrane potential of -80 mV. During the course of a 3 s application of L-glutamate (1 mM), half of the fibres responded with an initial peak showing fast inactivation, followed by a sustained component, while the other half of the fibres apparently lacked the initial peak. On average, the mean relative amplitude of the sustained component was 49 ± 8% of the initial peak one (n=23, mean ± S.E.M.). Responses to voltage ramps indicated that the sustained current component had a reversal potential around 0 mV. In another set of experiments, single action potentials or trains of action potentials overshooting to ~ +30 mV, were recorded in current-clamp mode in response to current steps from a membrane potential adjusted to -80 mV with slight basal current injection (on average, the resting potential of fibres before adjustment was -47 ± 5 mV, n=9). While the voltage-dependant sodium channel blocker tetrodotoxin (TTX, 1 µM) did not affect action potential responses, cadmium and lanthanum applied together (0.5 and 0.3 mM, respectively) induced disappearance of this all-or-none response. In voltage-clamp, the inward and outward currents underlying this action potential activity were recorded in response to controlled membrane depolarizations from a resting potential of -80 mV, in a physiological-like extracellular solution. In an extracellular solution devoid of sodium but containing 2 mM calcium and the potassium channel blockers tetraethylammonium and 4-aminopyridine, a current with an L-type calcium channel profile was recorded. This current was not affected by TTX, but was completely blocked by Cd2+-La3+. The activation threshold potential for this calcium current was ~ -40 mV and its mean maximal amplitude, reached at ~0 mV, was -8.5 ±1.9 A/F (n=13). Individual I-V curves were fitted with equation I(V)=Gmax(V-Vrev)/({1+exp[(V0.5-V)/k]}) and mean values for Gmax, Vrev, V0.5 and k were 217 ± 35 S/F, +39.8 ± 4.1 mV, -13.7 ± 1.8 mV and 5.4 ± 0.5 mV, respectively (n=13). This original cell preparation appears particularly suitable for patch-clamp studies and future investigations will allow a complete characterization of the pharmacological sensitivity of its membrane ionic currents as well as its calcium release apparatus properties, in order to explore excitation-contraction coupling in this insect model.
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Where applicable, experiments conform with Society ethical requirements.