Functional mapping of the N-terminal arginine cluster and C-terminal acidic residues of Kir6.2 channel fused to a G protein-coupled receptor

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.

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

“…These man-made LGICs, called ion channel-coupled receptors (ICCRs) broaden the properties of LGICs as: (1) they diversify the range of ligands to those recognized by the fused GPCRs, (2) they generate a sustainable signal, (3) they are selective to potassium, and (4) they have a basal activity that preserves a negative membrane resting potential and allows the detection of both activating and inhibiting ligands. The ICCR technology has multiple applications both in applied research as biosensors in interface with nano-electronic systems ( Lim et al., 2015 ) and in basic research for functional studies of ion channels ( Moreau et al., 2017 ; Principalli et al., 2017 ) and GPCRs ( Moreau et al., 2015 ; Niescierowicz et al., 2014 ).…”

“…While the molecular mechanism of this allosteric regulation is not precisely defined, structural evidence suggests that multiple domain-domain interactions are involved in the propagation of conformational changes from SUR ligand-binding sites to Kir6.2 gates ( Martin et al., 2019 ). Similarly, in ICCRs, both the N- and C-terminal domains of Kir6.2 play a role in the regulation by the fused GPCR ( Principalli et al., 2017 ).…”