The localization, protein interaction and cell surface mobility of neuronal alpha7 nicotine acetylcholine receptors

Abstract

In the brain, alpha7 neuronal nicotinic acetylcholine receptors (α7 nAChRs) have a special role amongst nAChRs. α7 nAChRs are forming homopentamers, they display a high permeability for Ca2+, and they are the most prevalent nAChRs in the brain. α7 nAChRs are found at the highest concentration in the hippocampus where they are located mostly on GABAergic interneurons and play an important role in learning and memory. Moreover α7 nAChRs have been involved in diseases such as Alzheimer’s disease (AD) and schizophrenia, and are attracting considerable scientific interest to elucidate their contribution to disease mechanisms. While central cholinergic circuits have been investigated very extensively, the cell- and molecular biological properties of α7 nAChRs have not been studied in depth. The exact subcellular localization of α7 nAChRs is still debated, in particular in relation to synaptic sites, and only two proteins interacting with α7 nAChR, namely RIC-3 and Src-family kinases, have been identified to date. However, none of them is involved in synaptic clustering of α7 nAChR. In chapter 2 we describe the discovery of PICK1 as a novel α7 nAChR interacting protein. Thereby the PDZ domain of PICK1 binds to the large cytoplasmatic loop of α7 nAChR. We present evidence that PICK1 regulates clustering of α7 nAChRs in rat hippocampal interneurons. The more detailed investigation of α7 nAChR clustering and surface dynamics demanded the ability to express exogenous gene constructs in cultured neurons. In chapter 3 we report an optimized transfection protocol for rat hippocampal neurons. Use of the magnetofection technique allowed the parallel transfection of several constructs and their expression in neurons for up to 3 weeks in vitro. To understand the clustering and localization of α7 nAChRs it is necessary to investigate the surface dynamics of single receptors. In chapter 4 we report a detailed analysis of α7 nAChR cell surface mobility, using α-BT and QDots labeled single receptor trafficking. α7 nAChRs were found to be very mobile within the membrane. Clusters were found to be mobility traps, suggesting α7 nAChRs interact with underlying scaffolding proteins at these sites. Mobility traps were found extrasynaptically and perisynaptically in close vicinity to GABAergic and glutamatergic postsynaptic densities. While extrasynaptic α7 nAChRs might activate Ca2+-dependent signaling pathways, the perisynaptic α7 nAChRs are probably playing a modulatory role in GABAergic and glutamatergic synaptic activity. α7 nAChR mobility was not only dependent on localization but also on chronic synaptic activity changes and activation of the receptor itself. Taken together, in this thesis work we identify α7 nAChR as a highly regulated receptor. The sites of α7 nAChR-dependent Ca2+ influx are tightly controlled by the cell. α7 nAChRs are clustered at distinct sites, reflecting functional heterogeneity. We identify for the first time a direct protein-protein interaction mechanism involved in the regulation of α7 nAChR clustering and possibly surface expression. We uncover α7 nAChR clusters as sites where mobility is constrained, but single receptors are able to diffuse in and out, confirming receptor clusters as steady-state receptor aggregations. We find α7 nAChR distributed all over the cell surface with clusters formed at extra and perisynaptic sites. We speculate that α7 nAChRs have a variety of different functions dependent on their localization. The distinct mechanisms of the particular α7 nAChR subpopulations remain unclear, and are left to be addressed in future work.