Engineering a Prokaryotic Cys-loop Receptor with a Third Functional Domain

Abstract

Background: Eukaryotic Cys-loop receptors contain three domains, whereas prokaryotic ones contain two because they lack the intracellular domain (ICD). Results: Functional chimeras were engineered by adding the serotonin receptor ICD to prokaryotic GLIC channels. Conclusion:The ICD is portable between eukaryotic and prokaryotic Cys-loop receptors. Significance: This portability will allow functional and structural studies of the ICD of diverse eukaryotic Cys-loop receptors.

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment

“…S5) with relatively large maximal currents typically close to 4 amps. A plot of whole cell currents versus proton concentration for the latter yielded a pH 50 for activation of 4.82 Ϯ 0.04 (Fig. 4B), consistent with that observed previously for oocyte-expressed GLIC (14).…”

“…The dose response of membrane-reconstituted GLIC incorporated into oocyte membranes is shifted to higher proton concentrations, suggesting a weaker ability of protons to gate open the channel. A lower pH 50 has also been observed in electrophysiology studies of GLIC reconstituted into asolectin membranes at high lipidprotein ratios. In fact, lipid effects may account for the altered pH 50 values observed for GLIC in different heterologous expression systems (49,50).…”

Desensitization Mechanism in Prokaryotic Ligand-gated Ion Channel

“…GLIC structures with propofol and desflurane reveal putative binding sites that are in agreement with functional measurements in nAChR (20). A functional chimera of GLIC-ECD with glycine receptor TMD yields anionic channels that are activated by pH (23), whereas a chimera of GLIC with the ICD of serotonin receptor functions as a pH-activated channel inhibited by chaperone protein RIC-3 (24). In both cases, the chimeras retain the functional properties of the individual domains, suggesting that the pathway for allosteric communication between the ECD-TMD-IMD underlying ligand binding and channel gating are essentially conserved across prokaryotic and eukaryotic channels.…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…Furthermore, their remarkable sensitivity to alcohols, general anesthetics, and other clinically relevant compounds (11,12) make them attractive targets for drug design. Remarkably, chimeras of GLIC with other eukaryotic members of the LGIC family retain the functional properties of the individual domains, strongly suggesting that the pathway for allosteric communication is essentially conserved across prokaryotic and eukaryotic channels (13,14).…”