A novel modulatory binding site for zinc on the GABAA receptor complex in cultured rat neurones.
1. The properties of gamma-aminobutyric acidA (GABAA) receptor-ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA-induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24-72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50% after 48.2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40-50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10-20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0.55% per day. 4. Activation of the GABA receptor by muscimol (0.2-2 microM) was also antagonized by zinc (50-100 microM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA-activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (tau 2, 2.1 ms) was unaffected by zinc, whereas the slow component time constant (tau 1, 21.7 ms) was slightly reduced to 17.1 ms. 8. The apparent single-channel conductance for GABA-activated ion channels was determined from the power spectra (gamma s = 22.7 pS) and also from the relationship between the mean GABA-induced inward current and the variance of the current (gamma v = 24 pS). Zinc (25-100 microM) did not affect the single-channel conductance. 9. Single GABA-activated ion channels were recorded from outside-out patches taken from the soma of large cerebellar neurones. Single GABA channels were capable of activation to multiple current amplitudes which were assessed into the following conductance levels: 8, 18, 23, 29 and 34 pS.(ABSTRACT TRUNCATED AT 400 WORDS)
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School of Pharmacy, Department of Pharmacology, London.
D000818:Animals; D001463:Barbiturates / Q000494:pharmacology; D001569:Benzodiazepines / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D016922:Cellular Senescence / Q000502:physiology; D005071:Evoked Potentials / Q000187:drug effects; D005260:Female; D018756:GABA Antagonists; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D001463:Barbiturates; D018756:GABA Antagonists; D007473:Ion Channels; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D015032:Zinc