Signal Transduction Pathways in the Pentameric Ligand-Gated Ion Channels

The mechanisms of allosteric action within pentameric ligand-gated ion channels (pLGICs) remain to be determined. Using crystallography, site-directed mutagenesis, and two-electrode voltage clamp measurements, we identified two functionally relevant sites in the extracellular (EC) domain of the bacterial pLGIC from Gloeobacter violaceus (GLIC). One site is at the C-loop region, where the NQN mutation (D91N, E177Q, and D178N) eliminated inter-subunit salt bridges in the open-channel GLIC structure and thereby shifted the channel activation to a higher agonist concentration. The other site is below the C-loop, where binding of the anesthetic ketamine inhibited GLIC currents in a concentration dependent manner. To understand how a perturbation signal in the EC domain, either resulting from the NQN mutation or ketamine binding, is transduced to the channel gate, we have used the Perturbation-based Markovian Transmission (PMT) model to determine dynamic responses of the GLIC channel and signaling pathways upon initial perturbations in the EC domain of GLIC. Despite the existence of many possible routes for the initial perturbation signal to reach the channel gate, the PMT model in combination with Yen's algorithm revealed that perturbation signals with the highest probability flow travel either via the β1-β2 loop or through pre-TM1. The β1-β2 loop occurs in either intra- or inter-subunit pathways, while pre-TM1 occurs exclusively in inter-subunit pathways. Residues involved in both types of pathways are well supported by previous experimental data on nAChR. The direct coupling between pre-TM1 and TM2 of the adjacent subunit adds new insight into the allosteric signaling mechanism in pLGICs. © 2013 Mowrey et al

β Subunit M2–M3 Loop Conformational Changes Are Uncoupled from α1 β Glycine Receptor Channel Gating: Implications for Human Hereditary Hyperekplexia

Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit