N-Aβcore attenuates the oAβ42-induced increase in α7β2-nAChR open-dwell time and total burst duration
Similar to homomeric α7-nAChR, heteromeric α7β2-nAChR open-dwell times within bursts were best fit with two components (Figure 5A; C1 and C2; see methods), indicating two distinct open-dwell time states. Similar analyses revealed a main effect of treatment on heteromeric α7β2-nAChR C1 mean open-dwell times within bursts (Figure 5B; F(7,68) = 14.4; P < 0.0001). Exposure to oAβ42 alone increases α7β2-nAChR open-dwell times (both C1 and C2) relative to effects of ACh alone, N-Aβ fragment alone, or N-Aβcore alone (P < 0.0001). Conversely, no significant differences in α7β2-nAChR single-channel open-dwell times were observed for C1 or C2 when exposed to N-Aβ fragment alone or N-Aβcore alone when compared to the application of ACh alone (P > 0.05).
However, compared to effects on α7β2-nAChR treated with oAβ42 alone, there were significant reductions in open-dwell time C1 in the presence of oAβ42 + N-Aβ fragment or oAβ42 + N-Aβcore (P < 0.0001). No differences were observed in C1 for α7β2-nAChR exposed to ACh alone, ACh + N-Aβ fragment, or ACh + N-Aβcore (P > 0.05). Moreover, single-channel C2 mean open-dwell times for α7β2-nAChR (Figure 5C) exposed to oAβ42 plus N-Aβcore are lower than those for exposure to oAβ42 alone (P < 0.0001). Effects of oAβ42 alone or in combination with N-Aβ fragment are indistinguishable (P > 0.05). However, both are significantly increased over the effects of N-Aβ fragment alone (P < 0.0001 and P < 0.001; respectively). By contrast, exposure to oAβ42 + N-Aβcore reduced α7β2-nAChR C2 mean open-dwell time relative to effects of oAβ42 alone and relative to what was observed for treatment with oAβ42 + N-Aβ fragment (P < 0.0001). Open-dwell times for α7β2-nAChR exposed to N-Aβcore (alone or in combination) were not significantly different to α7β2-nAChR treated with ACh alone (P > 0.05).
Application of oAβ42 alone increased α7β2-nAChR C1 and C2 single-channel open dwell-times within bursts compared to effects on α7-nAChR (P < 0.0001 and P < 0.01; respectively). Co-application of ACh + N-Aβ fragment increased C2 open-dwell time of α7-nAChR compared to that of α7β2-nAChR (Supplemental Figure S3D; P < 0.0001). Conversely, co-application of oAβ42+ N-Aβ fragment enhanced α7β2-nAChR C2 single-channel open dwell-time compared to the effects on α7-nAChR (P < 0.01). A main effect of receptor subtype was also observed for single-channel burst duration (Supplemental Figure S3F; F(1,144) = 19.4; P < 0.0001). Exposure to oAβ42 alone or in combination with N-Aβ fragment resulted in a significant enhancement in α7β2-nAChR total burst duration relative to the effects on α7-nAChR (P < 0.0001 and P < 0.01; respectively). Conversely, administration of N-Aβ fragment alone increased α7-nAChR burst duration compared to effects on α7β2-nAChR (P < 0.01). Together, these findings demonstrate the ability of oAβ42 to preferentially enhance α7β2-nAChR open-dwell times within bursts.
Further, total burst duration was enhanced for α7β2-nAChR when activated with oAβ42 alone when compared to exposure to ACh alone, N-Aβ fragment alone, or N-Aβcore alone (Figure 5D; P < 0.0001). Exposure to oAβ42 + N-Aβcore reduced α7β2-nAChR burst durations relative to the effects of oAβ42 alone (P < 0.0001) or relative to the effects of oAβ42 + N-Aβ fragment (P < 0.001). The effects on burst duration were indistinguishable for α7β2-nAChR exposed to oAβ42 alone or oAβ42 + N-Aβ fragment (P > 0.05). An analysis of single-channel open-dwell times revealed a main effect of receptor subtype on single-channel C1 and C2 open dwell-time between α7- and α7β2-nAChR across all treatment groups (Supplemental Figure S3C; F(1,140) = 4.9; P < 0.05 and Supplemental Figure S3D; F(1,140) = 3.6; P < 0.05; respectively).
As a control, an inactive amyloid core hexapeptide fragment (N-InAβcore; 1 µM) was empirically determined to be 1) inactive at α7- and α7β2-nAChR when applied alone and 2) ineffective at altering the single-channel properties of α7- and α7β2-nAChR single-channel kinetics when co-applied with ACh (compared to ACh alone; Supplemental Figure S4). Similar to the effects on α7β2-nAChR C1 and C2 open-dwell times, these findings demonstrate the ability of oAβ42 to enhance total burst duration, a feature unique to α7β2-containing nAChR. More importantly, these findings demonstrate the ability of the N-Aβcore hexapeptide to neutralize oAβ42 effects on α7β2-nAChR single-channel openings.