Research reportCa2+-sensitive inhibition by Pb2+ of α7-containing nicotinic acetylcholine receptors in hippocampal neurons
Introduction
The neurological effects of continuous exposure of humans to low levels of lead (Pb2+) represent a major concern in public health [10], [30]. Although this ubiquitous environmental pollutant at low levels does not induce obvious symptoms of poisoning, it causes long-lasting impairment of cognitive functions, especially early in development (reviewed in Refs. [6], [15]). Indeed, numerous epidemiological studies have reported that children are much more susceptible than adults to the deleterious neurological effects of Pb2+[26], [32], [38].
The effects of Pb2+ are unlikely to be accounted for by binding of this heavy metal to a single target. Pb2+ is known to modify the activity of a variety of Ca2+-binding proteins, including calmodulin [29], protein kinase C [33], Ca2+-ATPase [16], voltage-gated Ca2+ channels [8], Ca2+-activated K+ channels [37], and ligand-gated ion channels (reviewed in Refs. [12], [34]). Several lines of evidence indicate that on most protein targets the affinity of Ca2+-binding sites for Pb2+ is much higher than for Ca2+ itself (reviewed in [12], [31], [34]).
Among all ligand-gated ion channels studied to date, N-methyl-d-aspartate (NMDA)-type glutamate receptors and nicotinic acetylcholine receptors (nAChRs) bearing the α7 subunit, both of which have significant roles in cognitive functions (reviewed in Refs. [20], [23]), are the most sensitive to Pb2+; the IC50 for Pb2+ in inhibiting NMDA receptors ranges from 3 to 10 μM and the IC50 for Pb2+ in blocking α7 nAChR activity is around 10–20 μM [3], [13], [18], [19], [27], [28], [35]. Although the interaction of Pb2+ with NMDA receptors and α7-like nAChRs is non-competitive with respect to the agonists and is largely voltage independent [18], [24], [35], the exact mechanism(s) by which Pb2+ affects the activity of α7-like nAChRs remains unknown. Considering that both NMDA receptors and α7 nAChRs are sensitive to modulation by extracellular Ca2+[1], [21], and that Pb2+ and Ca2+ appear to compete for the same sites on NMDA receptors [24], it is possible that these cations also compete with one another for the same sites on α7 nAChRs. The major goals of the present study were (i) to investigate the kinetics of Pb2+’s interactions with α7 nAChRs; (ii) to determine whether blockade by Pb2+ of α7-like nAChRs is due to a direct interaction of Pb2+ with the receptors and/or to indirect actions of the heavy metal; and (iii) to evaluate interactions between Pb2+ and Ca2+ on α7 nAChRs. To this end, the whole-cell and the outside-out modes of the patch-clamp technique were applied to rat hippocampal neurons in culture.
Our present study demonstrates that Pb2+-induced blockade of α7 nAChRs has two components. One component has a rapid onset and is fully reversible upon washing of the neurons with Pb2+-free physiological solution. The other, which accounts for no more than 33% of the total inhibition by Pb2+ of the α7 nAChRs, develops more slowly and is only partially reversible upon washing of the neurons. Whereas the rapidly developing blockade by Pb2+ of the α7 nAChRs is likely due to the binding of the heavy metal to the extracellular domain of the receptor, the slowly developing blockade by Pb2+ of the α7 nAChRs appears to be associated with an intracellular action of the heavy metal. Evidence is also provided that the effect of Pb2+ on the α7-like nAChRs is Ca2+ dependent; the higher the extracellular concentration of Ca2+ the lower the effect of Pb2+.
Section snippets
Cultured hippocampal neurons
The primary cultures of neurons harvested from the hippocampus of 16–19-day-old rat fetuses were prepared as described previously [2]. Electrophysiological experiments were performed on hippocampal neurons cultured for 18–40 days.
Electrophysiological recordings
Transmembrane currents were recorded according to the standard patch-clamp technique [14] using a HEKA EPC-9 amplifier and the Pulse software (HEKA Electronic, Lambrecht, Germany). The resistance of borosilicate patch pipettes was 2–6 MΩ. To reduce the baseline noise
Pb2+ reduces the peak amplitude of α7 nAChR-mediated whole-cell currents
Approximately 85% of the hippocampal neurons in culture respond to acetylcholine (ACh) with fast-desensitizing currents that are sensitive to blockade by nanomolar concentrations of the α7 nAChR-antagonists α-bungarotoxin and methyllycaconitine, and are referred to as type IA currents [2]. Also, as reported earlier, choline, a precursor and a metabolite of ACh, acts as a full and selective agonist at the α7-like nAChRs in hippocampal neurons (reviewed in Ref. [1]); 10 mM choline is
Discussion
The results presented herein demonstrate that Pb2+ has a dual effect on α7 nAChRs: a rapidly developing inhibitory effect that is fully reversible, accounts for approximately 65–70% of the effect of Pb2+ on type IA currents, and is likely to be the result of a direct interaction of Pb2+ with the extracellular domain of the receptors, and a slowly developing inhibitory effect that is not reversible after removal of extracellular Pb2+ and may be due to an intracellular action of the heavy metal.
Acknowledgements
This study was support by United States Public Health Service Grant ES05730 (for E.X.A.), PRONEX 0888/96 (from Brazil, for E.X.A.), and Fogarty fellowship TW05389 (for A.M.). Part of this study was presented as an abstract in the 1999 Society for Neuroscience Meeting. The technical assistance of Ms. Mabel Zelle, Ms. Barbara Marrow, and Mr. Benjamin Cumming is also gratefully acknowledged.
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