| Summary: | 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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