Functional Mapping of the N-terminal Arginine Cluster and C-terminal Acidic Residues of Kir6.2 channel Fused to a G Protein-Coupled Receptor

Maria A. Principalli, Laura Lemel, Anaëlle Rongier, Anne-Claire Godet, Karla Langer, Jean Revilloud, Leonardo Darré, Carmen Domene, Michel Vivaudou, Christophe J. Moreau

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2 Citations (Scopus)
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Abstract

Ion channel-coupled receptors (ICCRs) are original man-made ligand-gated ion channels created by fusion of G protein-coupled receptors (GPCRs) to the inward-rectifier potassium channel Kir6.2. GPCR conformational changes induced by ligand binding are transduced into electrical current by the ion channel. This functional coupling is closely related to the length of the linker region formed by the GPCR C-terminus (C-ter) and Kir6.2 N-terminus (N-ter). Manipulating the GPCR C-ter length allows to finely tune the channel regulation, both in amplitude and sign (opening or closing Kir6.2). In this work, we demonstrate that the primary sequence of the channel N-terminal domain is an additional parameter for the functional coupling with GPCRs. As for all Kir channels, a cluster of basic residues is present in the N-terminal domain of Kir6.2 and is composed of 5 arginines which are proximal to the GPCR C-ter in the fusion proteins. Using a functional mapping approach, we demonstrate the role of specific arginines (R27 and R32) for the function of ICCRs, indicating that the position and not the cluster of positively-charged arginines is critical for the channel regulation by the GPCR. Following observations provided by molecular dynamics simulation, we explore the hypothesis of interaction of these arginines with acidic residues, and using site-directed mutagenesis, we identified aspartate D307 and glutamate E308 residues as critical for the function of ICCRs. These results demonstrate the critical role of the N-terminal and C-terminal charged residues of Kir6.2 for its allosteric regulation by the fused GPCR
Original languageEnglish
Pages (from-to)2144-2153
JournalBiochimica et Biophysica Acta (BBA)-Biomembranes
Volume1859
Issue number10
Early online date28 Jul 2017
DOIs
Publication statusPublished - Oct 2017

Keywords

  • ion channel-coupled receptor (ICCR)
  • protein engineering
  • electrophysiology
  • muscarinic receptor, molecular dynamics simulations

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