There have been several major accomplishments within the past fiscal year. The first is that we have discovered the presence of functional nAChR-mediated current responses from rat amygdala. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. Our results suggest possible mechanisms whereby nAChRs may be playing a significant role in fear and aversively-motivated memory.? ? Second, we utilized highly localized and rapid iontophoretic agonist delivery, combined with patch-clamp electrophysiology recordings and conventional fura-2 fluorescent imaging techniques, to compare the intracellular calcium levels in the soma and dendrites of rat hippocampal CA1 interneurons in the slice due to the activation of these alpha7 nAChRs. We found that in the dendrites, whereas the amplitudes of the current responses were smaller and the decay kinetics faster than the responses in the soma, the amplitudes of the calcium signals were significantly larger. Cultured hippocampal neurons were studied since the dendritic field lies in the same focal plane, which allowed for a broader investigation of the spatiotemporal dynamics of calcium signalling. In cultured neurons, the calcium signals in the dendrites were similar to those in slices. Interestingly in cultures, even though the amplitude of the alpha7 nAChR-mediated currents dramatically decreased with distance from the soma (from approximately 20-250 microm), the amplitude of the calcium signals did not correlate with distance. This indicates that the relative efficacy of alpha7 nAChR activation to increase calcium levels in dendrites increased severalfold with distance from the soma. These results may have implications for the role that alpha7 nAChRs have in regulating various signal transduction cascades, synaptic plasticity, and memory processes, via significant changes in calcium levels.? ? Third, we have continued to study the possible involvement of nAChRs relating to Alzheimers disease (AD) since dysfunctions in these receptors have been linked to AD, a human neurological disorder that is the leading cause of dementia. The most significant genetic risk factor for the development of Alzheimers disease is inheritance of the E4 allele of apolipoprotein E (apoE). Recent data has demonstrated the ability of apoE-derived peptides to inhibit nAChRs in rat hippocampus. We investigated the functional interaction between nAChRs and apoE-derived peptides in Xenopus oocytes expressing selected nAChRs. Both a 17 amino acid peptide fragment, apoE133-149, and an 8 amino acid peptide, apoE141-148, were able to maximally block ACh-mediated peak current responses for homomeric alpha7 nAChRs. ApoE peptide inhibition was dose-dependent, and voltage- and activity-independent. The current findings suggest that apoE peptides are non-competitive for acetylcholine and do not block functional alpha-bungarotoxin binding. Amino acid substitutions in the apoE peptide sequence suggest that the arginines are critical for peptide blockade of the alpha7 nAChR. Furthermore, mutation of tryptophan (W) 55 to alanine on the alpha7 nAChR blocked apoE peptide-induced inhibition of ACh-mediated alpha7 nAChR responses. Additional mutations at W55 suggest that hydrophobic interactions between the receptor and apoE141-148 are key for inhibition of alpha7 nAChR function. A mutated apoE peptide also demonstrated decreased inhibition at alpha7-W55A nAChRs as well as activity-dependent inhibition of both wildtype alpha7 nAChRs and alpha7-W55A receptors. Finally, a three-dimensional model of the alpha7 nAChR was developed based on the recently refined Torpedo nAChR. A structural model was proposed for the binding of apoE141-148 to the alpha7 nAChR where the peptide binds at the interface between two subunits, near the ACh binding site. Similar to the functional data, the computational docking suggests the importance of hydrophobic interactions between the alpha7 nAChR and the apoE peptide for inhibition of receptor function. The current data suggest a mode for apoE peptide binding that directly blocks alpha7 nAChR activity, and consequently may disrupt nAChR signaling.? ? Interestingly, the alpha7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype alpha7 nAChRs. This interpretation was validated by kinetic modeling of receptor function. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine, or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (alpha7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several charged (both positively and negatively) sulfhydryl reagents blocked ACh-induced responses for alpha7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat alpha7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions.
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