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On the kinetics of NMDA-associated ion channels : different agonists and the effects of anaesthetic drugs Sawyer, Dale Christopher


The majority of studies to date dealing with NMDAactivated ion channels have used whole-cell preparations to study macroscopic currents. The whole-cell current response to applied agonists consists of several components including the number of single channels activated by the agonist, the magnitude of the current passed by each ion channel, and the mean open time and frequency of opening of the ion channels. In order to determine the physiological and pharmacological properties of these individual components, it is useful to record single-channel currents through the NMDA ion channel. In this work I have used the cell-attached configuration of the patch clamp technique to study the effects of (a) different agonists and their stereoisomers and (b) intermediate and long-chain alcohols and volatile general anaesthetic agents on the single channel properties of the NMDA channel. An important point in this work is that intact cells, where channel gating from intracellular factors were preserved, were studied whereas the majority of single channel data involving NMDA channels has been obtained using outside-out patches, with little or no attempt to correlate such measurements with ion channel behaviour in the intact cell. I have studied the differences in mean open time and opening frequency (and thus probability of the channel being in the open state at a particular time) produced by different agonists of the NMDA channel; as well, the effect of agonist concentration on the frequency of channel opening was investigated. Cultured rat hippocampal CAl neurones were used in order to assess the single-channel kinetics of the stereoisomers of two different NMDA agonists, N-methylaspartic acid and homocysteic acid, as determined by on-cell patch-clamp recording with the agonists included in the pipette. In the absence of external Mg2+ the mean open time of NMDA channels, with all agonists, were diminished in an exponential manner with increasing patch hyperpolarization. No significant differences in conductance were observed between any of the agonists. However, significant differences in mean open time were found between the enantiomers NMDA and NMLA and between the enantiomers D- and L-homocysteic acid. Increasing the concentrations of the agonists in the patch pipette significantly increased the frequency of channel openings. These data on agonistinduced unitary currents can be correlated with whole-cell electrophysiological studies and autoradiographical binding affinity studies. Further studies were undertaken in order to assess the effects of several anaesthetic drugs, including the normal aliphatic alcohols butanol, pentanol, and octanol, as well as the volatile anaesthetics halothane and isoflurane, on the kinetics of unitary NMDA agonistactivated currents as recorded in the cell-attached configuration. While the channel conductance was not affected, mean open times were diminished at all patch potentials by the drugs; other effects, such as reduced frequency of channel opening, were also observed. Additional data, using the fluorescence probe fura-2 to illustrate the depressant effect of the n-alkanols on NMDAinduced increases in intracellular calcium, are provided in support of the results from the electrophysiological experiments. The information obtained in this study provides an explanation for such previous findings as the inhibition of whole-cell NMDA currents by n-alkanols and the attenuation of glutamate- and NMDA-stimulated intracellular calcium signals by volatile anaesthetics.

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