Multiple effects of tetraethylammonium on N-methyl-D-aspartate receptor-channels in mouse brain neurons in cell culture.
1. The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings. TEA (1-5 mM) decreased whole-cell NMDA responses. Kainate and quisqualate receptor-mediated responses were unaffected at these TEA concentrations. 2. In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA. Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition. In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased. These effects were not accompanied by any change in average channel open time. 3. Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized. The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential. The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV. The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA. 4. The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect. There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA. Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening. In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability. 5. NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels. Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)
Wright
J M
JM
Department of Pharmacology, NYSCVM, Cornell University, Ithaca 14853.
Kline
P A
PA
Nowak
L M
LM
D000818:Animals; D001921:Brain / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects*; D007608:Kainic Acid / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D016202:N-Methylaspartate / Q000037:antagonists & inhibitors*; D016318:Quisqualic Acid / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects*; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology*
D007473:Ion Channels; D011956:Receptors, Cell Surface; D013757:Tetraethylammonium Compounds; D016202:N-Methylaspartate; D019789:Tetraethylammonium; D016318:Quisqualic Acid; D007608:Kainic Acid; D005998:Glycine