Abstract
Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.
Highlights
Pentameric ligand-gated ion channels are neuroreceptors involved in fast synaptic transmission underlying the physiological processes of muscle action, gut activity, and neurological function
We found no difference between the WT and mutant simulation either at or above residue 441 on M4 from examining the local dynamical fluctuations of M4 by evaluating the root mean square fluctuations (RMSF) of each residue (Figure 2)
The aim of this work was to probe the effects of the M4 helix in 5-HT3A receptor (5-HT3AR) function using the non-functional Y441A mutation
Summary
Pentameric ligand-gated ion channels (pLGICs) are neuroreceptors involved in fast synaptic transmission underlying the physiological processes of muscle action, gut activity, and neurological function. They are present throughout the central and peripheral nervous systems, and mediate the action of biologically active compounds including nicotine, alcohol, and many anesthetics (Nemecz et al, 2016). While this helix is fundamentally an amphipathic barrier to the hydrophobic lipid environment, there is growing evidence that the M4 helix plays a key role in pLGIC function: mutations in the M4 helices of mammalian pLGICs have been shown to reduce or inhibit channel opening (Cory-Wright et al, 2018; Tang et al, 2018; Mesoy et al, 2019), or promote channel function (da Costa Couto et al, 2020) the exact mechanism is not yet clear
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