Drug interactions with voltage-dependent K channels and N-methyl-D-aspartate (NMDA) receptor coupled cation channels were studied in cultured hippocampal neurons using whole-cell voltage-clamp and single channel recording techniques.
The aim of this work was to develop new strategies for the rational development of antiepileptic drugs based upon their interaction with neuronal ion channel systems. Work was focused in three areas: (1) phencyclidine (PCP) related drugs, (2) antagonists of the glycine modulatory site on the NMDA receptor, and (3) K channel activator drugs. PCP (0.5 - 1000 muM) caused a reduction in the maximum conductance of the slowly activating K current I-K in hippocampal neurons [IC-50(+30 mV), 22 muM] without altering its voltage-dependency. The PCP block of I-K diminished at depolarized potentials. Analysis according to the scheme of Woodhull suggested that block occurs via binding to an acceptor site (presumably within the channel pore) that senses 40-50% of the transmembrane electrostatic field. PCP reduced inward current responses induced by the excitatory amino acid agonist NMDA at substantially lower concentrations than those required for its effects on K channels [IC-50(60 mV), 0.45 muM]. The affinity of PCP for its acceptor site on I-K channels is 13 times lower than its affinity for NMDA-receptor associated channels. Therefore, at low doses, the behavioral effects of the drug are more likely to result from an interaction with NMDA receptor-channels than voltage-dependent K channels. Another approach to diminishing NMDA-receptor mediated excitation is blockade of the glycine modulatory site on the NMDA-receptor channel complex. Using the whole-cell voltage-clamp technique, we demonstrated that the glycine-analog cycloleucine is a competitive antagonist at the glycine site. In the presence of 1 muM glycine, cycloleucine caused a reversible, dose-dependent inhibition of NMDA responses with an IC-50 of 24 muM. An increase in glycine to 100 muM resulted in a shift to the right of the cycloleucine concentration-effect curve (IC-50, 1.4 muM). Cycloleucine failed to affect kainic acid and quisqualic acid evoked currents at concentrations which inhibited NMDA responses. As an alternative anticonvulsant strategy, we explored means of pharmacologically activating voltage-dependent K channels. We observed that the antihypertensive cromakalim (BRL 34915) causes a dramatic increase in the slowly activating, sustained outward current in cultured hippocampal neurons. Drugs like cromakalim which activate K channels in CNS neurons have potential as anticonvulsants.
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