Lana, B; (2013) The voltage-gated calcium channel α₂δ-1 subunit: splice variants and interacting proteins. Doctoral thesis , UCL (University College London).

Abstract

á2ä-1 is an auxiliary subunit of the voltage-gated calcium channels, and it regulates the trafficking and functions of the channel complex. á2ä-1 subunits contain the binding site for gabapentin (GBP), used for the treatment of neuropathic pain. The first aim of this thesis was to determine whether important functional properties of á2ä-1, such as the ability to regulate calcium currents and binding to GBP, were altered by alternative splicing events on á2ä-1. In addition, the distribution of á2ä-1 splice variants in dorsal root ganglia (DRGs) of naive and spinal nerve ligated (SNL) rats was analysed by RT-PCR and gel electrophoresis. The results obtained from [3H]-GBP binding experiments identified two splice variants characterised by a low binding affinity to GBP. One of those, which is known to undergo up-regulation in DRGs neurons upon SNL, was found to be specifically expressed in smaller neurons, which are normally nociceptive. The interaction of endogenous ligands with á2ä-1 can also contribute to influence the the binding affinity of GBP to á2ä-1. Therefore, the other goal of this study was to analyse the interaction of endogenous ligands with á2ä-1. First, co-immunoprecipitation and radioligand binding experiments demonstrate that á2ä-1 interacts with thrombospondins (secreted proteins that bind á2ä-1 and promote synaptogenesis) and that this interaction reduces the binding affinity of GBP to á2ä-1. Secondly, it was investigated wheather another protein: the low-density lipoprotein receptor (LDLR)-related protein-1 (LRP1) could also interact with á2ä-1 and TSP. It was found that LRP1 co-immunoprecipitates with TSP4 and it also decreases the amount of GBP binding to á2ä-1, suggesting that LRP1 might be involved in promoting the interaction between á2ä-1 and TSP4. These studies have shown that the existence of alternative splice variants of á2ä-1 and the interaction of á2ä-1 with endogenous interactors like TSPs and LRP1 affect the functionality of these subunits. An altered functionality might play an important role upon nerve damage, when á2ä-1 becomes up-regulated, because GBP requires binding to á2ä-1 to exert its therapeutic effect

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