Transducer properties of a mechanoreceptor : an electrophysiological and pharmacological study of the crayfish stretch receptor

Sammanfattning: The abdominal stretch receptor organ of the crayfish, an analog to the human muscle spindle, contains one slowly adapting (SA) and one rapidly adapting (RA) receptor neuron. The general aims of this project were to elucidate, in the SA and RA neurons, the participation of voltagegated Na+ and K+ channels in overall transducer process and adaptation, and to study possible blocking agents specific to mechanosensitive (MS) channels. The two-electrode current and voltage clamp techniques were used to record whole cell membrane potential and current; macropatch and single channel recordings were made using the patch clamp technique after the neuronal membrane was enzymatically freed from covering glial cells. In the SA neuron at least two populations of Na+ channels may be present in the soma, axon hillock and axon. In the RA neuron the majority of Na+ channels were found in axon about 250 gin away from the soma, and only one population of Na+ channels seemed to be present. In SA neuron, voltage gated Na+ and K+ channels were more concentrated in the part of the soma close to the axon hillock, and their current densities were decreased towards the dendrites. A low conductance (13 pS) outward rectifying, voltage gated K+ channel was characterized in the SA neuron. It was activated by depolarization of > 20 mV, and its single channel current amplitude and open probability were voltage dependent. The averaged current showed a pronounced delay in activation with little inactivation within 80 ms. At least two closed states preceded two open states. This 13 pS delayed rectifier is important in the repolarization of the action potentials. These results suggest that the action potential initiation site differs in SA and RA neurons and that the spatial distribution is an important factor in the difference between their excitable and adaptive properties. The effects on the transducer properties of four local anesthetics (lidocaine, its meta-isomer LL33, tetracaine and bupivacaine) and a calmodulin antagonist, CGS 9343B were investigated in SA neurons. Lidocaine reversibly increased the receptor current by a major K+ conductance increase through the MS channels, whereas LL33, bupivacaine and tetracaine reversibly reduced the current in a dose-dependent way without changing the passive membrane conductance. CGS 9343B reversibly blocked the receptor current in a voltage- and dosedependent manner (Kd = 26.8 [my]M). The block was stimulus-independent, and extracellular Ca2+ had no effect on it. The local anesthetic effects, whether activating or blocking MS channels, were correlated to the oil:water distribution coefficients. CGS 9343B, in addition to accumulating in the lipid membrane, may also interact with MS channel proteins. In summary, these studies indicate that several kinds of voltage-gated Na+ and K+ channels are present in SA and RA neurons, and their differing relative occurrence and spatial distribution suggest that these channels play important roles in the generation of impulses and adaptation behaviours in the SA and RA neurons. The four local anesthetics and the calmodulin antagonist can either activate or block the NIS channels in the dendrites of the receptor neuron probably by interference with the lipid phase, yielding useful information about the properties of the MS channels.