The Role of Intracellular Linkers in Gating and Desensitization of Human Pentameric Ligand-Gated Ion Channels

It has recently been proposed that post-translational modification of not only the M3-M4 linker but also the M1-M2 linker of pentameric ligand-gated ion channels modulates function in vivo. To estimate the involvement of the M1-M2 linker in gating and desensitization, we engineered a series of mutations to this linker of the human adult-muscle acetylcholine receptor (AChR), the alpha 3 beta 4 AChR and the homomeric alpha 1 glycine receptor (GlyR). All tested M1-M2 linker mutations had little effect on the kinetics of deactivation or desensitization compared with the effects of mutations to the M2 alpha-helix or the extracellular M2-M3 linker. However, when the effects of mutations were assessed with 50 Hz trains of similar to 1ms pulses of saturating neurotransmitter, some mutations led to much more, and others to much less, peak-current depression than observed for the wild-type channels, suggesting that these mutations could affect the fidelity of fast synaptic transmission. Nevertheless, no mutation to this linker could mimic the irreversible loss of responsiveness reported to result from the oxidation of the M1-M2 linker cysteines of the alpha 3 AChR subunit. We also replaced the M3-M4 linker of the alpha 1 GlyR with much shorter peptides and found that none of these extensive changes affects channel deactivation strongly or reduces the marked variability in desensitization kinetics that characterizes the wild-type channel. However, we found that these large mutations to the M3-M4 linker can have pronounced effects on desensitization kinetics, supporting the notion that its post-translational modification could indeed modulate alpha 1 GlyR behavior.