The role of charged residues in independent glycine receptor folding domains for intermolecular interactions and ion channel function

Glycine receptor (GlyR) truncations in the intracellular TM3-4 loop, documented in patients suffering from hyperekplexia and in the mouse mutant oscillator, lead to non-functionality of GlyRs. The missing part that contains the TM3-4 loop, TM4 and C-terminal sequences is essential for pentameric receptor arrangements. In vitro co-expressions of GlyRa1-truncated Ndomains and C-domains were able to restore ion channel function. An ionic interaction between both domains was hypothesized as the underlying mechanism. Here, we analysed the proposed ionic interaction between GlyR N-and Cdomains using C-terminal constructs with either positively or negatively charged N-termini. Charged residues at the Nterminus of the C-domain did interfere with receptor surface expression and ion channel function. In particular, presence of negatively charged residues at the N-terminus led to significantly decreased ion channel function. Presence of positive charges resulted in reduced maximal currents possibly as a result of repulsion of both domains. If the C-domain was tagged by a myc-epitope, low maximal current amplitudes were detected. Intrinsic charges of the myc-epitope and charged N-terminal ends of the C-domain most probably induce intramolecular interactions. These interactions might hinder the close proximity of C-domains and N-domains, which is a prerequisite for functional ion channel configurations. The remaining basic subdomains close to TM3 and 4 were sufficient for domain complementation and functional ion channel formation. Thus, these basic subdomains forming ahelical elements or an intracellular portal represent attractants for incoming negatively charged chloride ions and interact with the phospholipids thereby stabilizing the GlyR in a conformation that allows ion channel opening.