P1-169: allosteric regulation of α7 nachrs by low and high aβ(1-42) concentrations

Several studies (including our own) have found that high nM-μM concentrations of Aβ peptides inhibit α7 nAChR activity. In contrast, recent studies show that picomolar concentrations interact with α7Rs in a facilitatory way, suggesting potentially positive roles for Aβ in certain learning- and memory-related processes (Puzzo et al., 2008). The present study aims to characterize the nature of this facilitory interaction. Mouse α7 nAChR subunit cRNA was synthesized from mα7 nAChR/pGEM-HE plasmid DNA and microinjected into Xenopus laevis oocytes. Functional expression of mouse α7Rs was evaluated fourth day post injection via two electrode voltage clamp of microinjected oocytes clamped at -60mV and exposed to a saturating concentration (500μM) of acetylcholine (ACh). 2-4hr exposure to 100pM Aβ(42) increased peak amplitudes and slowed desensitization of ACh-evoked α7R currents expressed in oocytes similar to, but less strongly than, the type II positive allosteric modulator (PAM) PNU 120596. PNU 120596 (but not the type I PAM, genistein) also occluded the effects of 100pM Aβ(1-42), suggesting that 100pM Aβ acts as a type II PAM at α7Rs. Analysis of ACh-induced currents of the M276L mutant in the presence of 100pM Aβ or 15uM PNU 120596 showed the expected reduction in PNU facilitation (Young et al, 2008) and complete elimination of 100pM Aβ facilitation. A different mutant (M283L) that's critical for type I PAM sensitivity showed the expected reduction in facilitation by Genistein; tests of Aβ concentrations are currently in progress. Surprisingly, higher concentrations of Aβ(1-42) [100 and 500 nM] that partially suppressed ACh-evoked currents through wt α7 Rs failed to suppress M276L α7Rs. Instead, moderate but non-significant facilitations of peak and total currents were observed, as was a small slowing of desensitization. Our results for M276L α7Rs suggest that 100pM Aβ(42) does in fact modulate α7R activity through conventional type II PAM action. However, analysis of the effects of 100pM Aβ(42) on M283L is necessary to rule out the possibility of a type I PAM-like interaction. The observation that both facilitatory and inhibitory Aβ(1-42) concentrations were attenuated by the M276L mutation suggests a shared or overlapping (rather than distinct) binding site.