The inhibition of single N-methyl-D-aspartate-activated channels by zinc ions on cultured rat neurones.
1. Single channels activated by N-methyl-D-aspartate (NMDA) were studied in outside-out patches of cultured hippocampal neurones in the presence of glycine and absence of magnesium. The effects of the transition metal ions zinc and cadmium on NMDA channels were tested by placing the membrane patch at the mouth of one of an array of large barrelled flow pipes. 2. Amplitude histograms revealed several conductance levels between 5 and 45 pS with the majority of NMDA-activated openings greater than 25 pS. Zinc (5-100 microM) and cadmium (30-100 microM) reduced the number of large conductance events in a voltage-independent manner. Zinc (30 microM) reduced the large conductance openings by approximately 70-80%. The small number of events under 20 pS precluded quantitative assessment of the effects of zinc and cadmium on these conductance levels. Zinc inhibition of the calculated macroscopic current due to NMDA-activated channels could be fitted with a single binding site isotherm with an IC50 of 12 microM. 3. Zinc and cadmium also reduced the mean open time of the two largest conductance events of 38 and 43 pS; this reduction was voltage independent. Open-time histograms were fitted with the sum of two exponentials. In the presence of 5 microM-NMDA at -60 mV, tau o2 = 10.49 ms and tau o1 = 1.47 ms; in 30 microM-zinc, tau o2 = 3.49 and tau o1 = 0.8 ms. The 'blocking' rate constant calculated at a membrane potential of +40 mV from the slope of 1/tau o2 vs. [zinc]o was 4 x 10(6) M-1 S-1. 4. Closed-time analysis revealed brief (tau c = 0.4-1.0 ms) zinc-insensitive gaps; longer closed-time intervals were not analysed since all patches contained more than one channel. Both burst duration and the number of bursts were reduced in the presence of zinc. 5. At holding potentials negative to -40 mV in magnesium-free solutions, zinc also induced high-frequency flickering of the open channel which included complete channel closures at 4 kHz filtering. No zinc-induced flickering was seen at positive membrane potentials. The flickering was dose dependent, becoming prominent at zinc concentrations above 30 microM. Cadmium did not induce flickering at concentrations up to 100 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Vollum Institute, Oregon Health Sciences University, Portland 97201.
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Westbrook
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002104:Cadmium / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006624:Hippocampus / Q000502:physiology*; D007473:Ion Channels / Q000187:drug effects*; D016202:N-Methylaspartate / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology*
D007473:Ion Channels; D002104:Cadmium; D016202:N-Methylaspartate; D015032:Zinc