Schönrock M et al. (2019), Coupling of a viral K+-channel with a glutamate...

XB-ART-56920

Commun Biol 2019 Jan 01;2:75. doi: 10.1038/s42003-019-0320-y.

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Coupling of a viral K+-channel with a glutamate-binding-domain highlights the modular design of ionotropic glutamate-receptors.

Schönrock M , Thiel G , Laube B .

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Ionotropic glutamate receptors (iGluRs) mediate excitatory neuronal signaling in the mammalian CNS. These receptors are critically involved in diverse physiological processes; including learning and memory formation, as well as neuronal damage associated with neurological diseases. Based on partial sequence and structural similarities, these complex cation-permeable iGluRs are thought to descend from simple bacterial proteins emerging from a fusion of a substrate binding protein (SBP) and an inverted potassium (K+)-channel. Here, we fuse the pore module of the viral K+-channel KcvATCV-1 to the isolated glutamate-binding domain of the mammalian iGluR subunit GluA1 which is structural homolog to SBPs. The resulting chimera (GluATCV*) is functional and displays the ligand recognition characteristics of GluA1 and the K+-selectivity of KcvATCV-1. These results are consistent with a conserved activation mechanism between a glutamate-binding domain and the pore-module of a K+-channel and support the expected phylogenetic link between the two protein families.

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Species referenced: Xenopus laevis
Genes referenced: fubp1

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	Fig. 1. Design of KcvATCV-1/GluA1chimeras. a The cartoon depicting the topology of a subunit of the KcvATCV-1 (blue), GluA1 (brown), and the chimera GluATCV harboring the membrane-spanning domain of the KcvATCV-1. Amino-terminal domain (NTD), ligand-binding domain (LBD), C- terminal domain (CTD), pore helix (P), transmembrane domains (TM respectively M in case of GluA1), substrate-binding-protein (SBP, dark brown). Permeant cations are indicated. b Structural overlay TM of KcvATCV-1 and GluA2. TM1 and pore helix as well as M1 and M2 of GluA2 are transparent. TM of KcvATCV-1 (TM2) and GluA2 (M3) are in full color. Helices were superimposed by aligning main-chain atoms of TM2 segments from KcvATCV-1 model (see Methods) on crystal structure of M3 domain of GluA2 subunit (Protein Database entry 3KG220) (side view). Backbones and residues of subunits are illustrated in ribbon representations (blue: KcvATCV-1; brown: GluA2) within iGluRs conserved SYTANLAAF region (green) and position of G77 and F78 of KcvATCV-1 (red). c Design of GluATCV constructs. Partial sequence alignment of GluA1, GluA2, and different GluATCV constructs. Illustration of amino acid cutting sites of different GluA1/KcvATCV-1 chimeras (GluATCV). Chimeras harboring different lengths of the SYTANLAAF motif (green) with corresponding residue numbering of mature subunits (KcvATCV-1 blue; GluA1 brown) are indicated. GluATCVlong (linker length + 13 aa); GluATCVshort (linker length + 8 aa), and GluATCV (no linker). Residues found at cutting sites and within the SYTANLAAF motif are highlighted. Secondary structure elements found are illustrated above the sequence. d Functional characterization of chimeric GluATCV constructs. Representative whole-cell currents of glutamate (Glu) responses and Ba2+ inhibition of GluA1/KcvATCV-1 chimeras upon heterologous expression in Xenopus oocytes recorded at −70 mV membrane potential. Oocytes expressing GluATCVshort and GluATCV were superfused with the indicated concentration of glutamate in the absence and presence of 500 µM Ba2+. Current traces illustrating inhibitory effects of Ba2+ in both chimera. Note that K+-specific blocker Ba2+ inhibits both glutamate-induced currents elicited from GluATCV and leak current in GluATCVshort expressing oocytes. Black dotted line indicates Ba2+ insensitive leakage current. Bars show timepoint and duration of the application. Gray dotted line and arrows indicate glutamate induced current (IGlu) and barium blockable current (IBa)
	Fig. 2. Increase of Glu-gating efficiency in GluATCV by TM2 point mutations. a Single channel recordings of the KcvATCV-1 and KcvATCv-1. Currents of KcvATCV-1 and KcvATCV-1 (mutated at aa positions (G77S, F78L)) were recorded at a membrane potential of +60 and −60 mV upon reconstitution in planar lipid bilayer (see Methods). Note the difference of the open probability in the characteristic single channel fluctuations of the two K+ channels. b Analysis of the open probability and single channel conductance of KcvATCV-1 and KcvATCV-1. Plot of the open probabilities (Po) and single channel conductance (pS) of the wt and mutant KcvATCV-1 channel by calculating the time of occupancy of the open state (O) and the closed state (C) from 4 independent 1 min recordings at +60 and −60 mV (p < 0.001; unpaired two-side t-test; n = 4). c Overlay of representative recordings of glutamate (Glu) responses and Ba2+ inhibition at GluATCV (red) and GluATCV (black). Arrow illustrates the differences in the ratio of the inhibition of the glutamate-induced currents and the resting leakage by the K+-specific blocker Ba2+ in GluATCV and GluATCV* expressing oocytes. Dotted line indicates the Ba2+ insensitive leakage current. d Fractional contribution of the Ba2+-sensitive leakage- and of Glu-induced currents in GluATCV constructs. Percentage of the Glu-induced currents of the total Ba2+-sensitive current in GluATCV* is highly significant increased compared to the GluATCV (68.2 ± 5.2% vs. 36.4 ± 4.6%, respectively; p = 0.0017; unpaired two-side t-test; 95% confidence interval = −47.74 to −15.94; n = 5)
	Fig. 3. Functional characterization of the GluATCV chimera. a Glu responses of GluA1 and GluATCV* upon expression in Xenopus oocytes. Traces of Glu responses of GluA1 and GluATCV* with different concentrations of glutamate. The bars show the duration of the application of the corresponding concentration. b Glutamate dose–response curves recorded from GluA1 (squares) or GluATCV* (circles) expressing oocytes. Both show a similar EC50 value of 5.8 ± 1.2 and 4.0 ± 0.4 µM for GluATCV* and GluA1, respectively. c CNQX-inhibition curve of GluA1 and GluATCV* at the EC50 value of glutamate. The CNQX IC50 is 4.4 ± 0.9 and 2.0 ± 0.1 µM for GluATCV* and GluA1, respectively. d Plot of the reversal voltages of the GluA1 and GluATCV* receptors against the extracellular K+ concentration. Reversal voltages were estimated by current–voltage (I–V) recordings in different ringer solutions containing 10, 50, 100, and 150 mM K+. The proportional shift of the reversal voltage as a function of the concentration of K+ in the extracellular medium with a slope of 59.3 ± 4.9 for the GluATCV* and 3.9 ± 4.7 for GluA1 confirms a high selectivity for K+ over Na+ in the GluATCV* channel
	Fig. 4. Design of minimal Glu-gated viral potassium channels. a Schematic drawings and functional expression of the deletion and mutant constructs used. Cartoons depicting the NTD- and the M4 truncated versions and the cysteines mutated (indicated by red stars); Inset: Point mutations in the LBD (N407C) and TM1 segment (V152C) thought to form a disulfide-bridge are highlighted in red based on our homology model against 3KG2. Numbers correspond to amino acid positions in the mature protein. Imax currents of each mutant, with and without DTT in the case of the cysteine double mutant, are shown (p = 0.8; one-way ANOVA with turkey correction; n = 4–10). b Overlay of representative whole cell current traces of Glu-gated GluATCV, GluATCVΔNTD ΔM4, and GluA1. The respective traces in response to application of 100 µM glutamate (Glu) are shown in magenta, black, and orange, respectively. Note that the overall shape of the glutamate-induced whole cell currents is similar between the constructs. c Glu dose–response curves of GluATCV* (black circles), the deletion mutant GluATCVΔNTD ΔM4 (magenta squares), and the double mutant GluATCVΔNTD ΔM4 V152/N402C (green triangles) without DTT (open symbols) and in the presence of 5 mM DTT (closed symbols). EC50 GluATCV* 5.8 ± 1.1 µM (−DTT) and 5.1 ± 1.1 µM (+DTT); GluATCVΔNTD ΔM4 20.8 ± 2.6 µM (−DTT) and 22.6 ± 2.9 µM (+DTT); GluATCVΔNTD ΔM4 V152/N402C 5.9 ± 0.4 µM (−DTT) and 10.5 ± 0.7 µM (+ DTT). Note that, in contrast to GluATCV* and GluATCVΔNTD ΔM4, only the GluATCVΔNTD ΔM4 V152/N402C shows a significant changed EC50 value in the presence of DTT (green closed triangles) indicative of an intrasubunit disulfide link between LBD and TM1

References [+] :

Amin, Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors. 2017, Pubmed