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

Raman Goyal,Michaela Jansen,Ahmed Abdullah Salahudeen

doi:10.1074/jbc.m111.269647

Abstract

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Highlights

Eukaryotic Cys-loop receptors contain three domains, whereas prokaryotic ones contain two because they lack the intracellular domain (ICD)
We have shown previously that the 5-HT3A-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT3A receptors in X. laevis oocytes
All chimeras contain the complete 5-HT3AICD but differ in the number of amino acids contributed by the GLIC M3M4 linker and its flanking regions both N-terminal and C-terminal to the 5-HT3A-ICD insertion (Fig. 1, B and C)

Summary

Introduction

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. We added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT3A) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We have shown previously that the 5-HT3A-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT3A receptors in X. laevis oocytes. Expression of both functional 5-HT3A chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT3A-ICD in the chimeras.

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Conclusion