Manville RW and Abbott GW (2020), Potassium channels act as chemosensors for solu...

XB-ART-58223

Commun Biol 2020 Feb 28;31:90. doi: 10.1038/s42003-020-0820-9.

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Potassium channels act as chemosensors for solute transporters.

Manville RW , Abbott GW .

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Potassium channels form physical complexes with solute transporters in vivo, yet little is known about their range of possible signaling modalities and the underlying mechanisms. The KCNQ2/3 potassium channel, which generates neuronal M-current, is voltage-gated and its activity is also stimulated by binding of various small molecules. KCNQ2/3 forms reciprocally regulating complexes with sodium-coupled myo-inositol transporters (SMITs) in mammalian neurons. Here, we report that the neurotransmitter γ-aminobutyric acid (GABA) and other small molecules directly regulate myo-inositol transport in rat dorsal root ganglia, and by human SMIT1-KCNQ2/3 complexes in vitro, by inducing a distinct KCNQ2/3 pore conformation. Reciprocally, SMIT1 tunes KCNQ2/3 sensing of GABA and related metabolites. Ion permeation and mutagenesis studies suggest that SMIT1 and GABA similarly alter KCNQ2/3 pore conformation but via different KCNQ subunits and molecular mechanisms. KCNQ channels therefore act as chemosensors to enable co-assembled myo-inositol transporters to respond to diverse stimuli including neurotransmitters, metabolites and drugs.

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Species referenced: Xenopus laevis
Genes referenced: slc5a3

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	Fig. 1. GABA regulates SMIT1 activity via co-assembled KCNQ2/3.All error bars in the study indicate SEM. All n values in the study represent biologically independent samples or experiments. a Topology of KCNQ channels (two of four subunits shown) and an exemplar of the SLC5A solute transporter family, vSGLT, which may reflect SMIT topology. b Effect of GABA (100 µM) on [3H]Myo-inositol uptake by whole dorsal root ganglia (n = 10) isolated from postnatal day 8 rats. CPM = counts per minute. c Effects of GABA (100 µM; n = 17) on [3H]Myo-inositol uptake by SMIT1 (n = 29 without GABA) expressed alone in Xenopus oocytes. Control oocytes (black) were injected with water instead of SMIT1 cRNA (n = 16). d Effects of small molecules as indicated (100 µM) on [3H]Myo-inositol uptake by SMIT1 co-expressed with KCNQ2/KCNQ3 subunits in Xenopus oocytes ([3H]Myo-inositol n = 41; Glutamate n = 11; GABA n = 27; BHB n = 26; GABOB n = 23; Retigabine n = 15). e Effects of small molecules as indicated (100 µM) on [3H]Myo-inositol uptake by SMIT1 co-expressed with KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) subunits in Xenopus oocytes ([3H]Myo-inositol n = 14; GABA n = 25; BHB n = 18; GABOB n = 28).
	Fig. 2. SMIT1 alters effects of GABA on KCNQ2/KCNQ3 channels.All error bars indicate SEM. a Mean traces for KCNQ2/KCNQ3 (Q2/Q3) co-expressed with SMIT1 (S1) in the absence (Control) or presence of GABA (100 µM) (n = 5). Voltage protocol for this and all similar recordings in this study, inset. Structure and electrostatic surface potential heatmap plots shown for GABA. Red, negative charge, blue, positive charge for all heatmaps in this article. b Mean tail current (at arrow in (a)) and normalized tail current (G/Gmax) versus prepulse voltage for KCNQ2/KCNQ3–SMIT1 traces as in (a) (n = 5). c GABA effects on KCNQ2/KCNQ3–SMIT1 activation time constant = τact (n = 5). d Voltage dependence and dose response (at −60 mV) for GABA effects on KCNQ2/KCNQ3 (red; n = 5) and KCNQ2/KCNQ3–SMIT1 (magenta; n = 6) current. e GABA dose response for effects on KCNQ2/KCNQ3 (red; n = 5) and KCNQ2/KCNQ3–SMIT1 (magenta; n = 6) activity (shift in midpoint voltage of activation). f Mean traces for KCNQ2/KCNQ3 (Q2/Q3) co-expressed with SMIT1 (S1) in the absence (control) or presence of BHB (100 µM) (n = 5). g Mean and normalized tail current (G/Gmax) versus prepulse voltage for KCNQ2/KCNQ3–SMIT1 traces as in (f) (n = 5). h BHB effects on KCNQ2/KCNQ3–SMIT1 activation rate (n = 5). i Voltage dependence and dose response (at −60 mV) for BHB effects on KCNQ2/KCNQ3 (red) and KCNQ2/KCNQ3–SMIT1 (blue) (n = 5). j BHB dose response for effects on KCNQ2/KCNQ3 (red) and KCNQ2/KCNQ3–SMIT1 (blue) activity (shift in midpoint voltage of activation) (n = 5). k Mean traces for KCNQ2/KCNQ3 (Q2/Q3) co-expressed with SMIT1 (S1) in the absence (control) or presence of GABOB (100 µM) (n = 5). l Mean and normalized tail current (G/Gmax) versus prepulse voltage for KCNQ2/KCNQ3–SMIT1 traces as in (k) (n = 5). m GABOB effects on KCNQ2/KCNQ3–SMIT1 activation rate (n = 5). n Voltage dependence and dose response (at −60 mV) for GABOB effects on KCNQ2/KCNQ3 (blue; n = 5) and KCNQ2/KCNQ3–SMIT1 (orange; n = 6). o GABOB dose response for effects on KCNQ2/KCNQ3 (blue; n = 5) and KCNQ2/KCNQ3–SMIT1 (orange; n = 6) activity (shift in midpoint voltage of activation).
	Fig. 3. GABA increases relative sodium and cesium permeability of KCNQ channels that it activates.All error bars indicate SEM; n values indicate biologically independent experiments. a Representative traces from recordings of Xenopus laevis oocytes injected with cRNA encoding KCNQ2/3 in 100 mM K+ (voltage protocol inset). b Mean current–voltage relationship for KCNQ2/3 in 100 mM K+ (black), Cs+ (blue), Rb+ (red) and Na+ (magenta; n = 5), in the absence or presence of GABA (100 µM; n = 6) as indicated, values quantified from arrow in panel (a). c Estimated mean ion permeability relative to that of K+ versus ionic radius (Pauling) through KCNQ2/Q3 in the absence (black; n = 5) and presence (purple; n = 6) of GABA (100 µM). p < 0.05. d Representative traces from recordings of oocytes injected with cRNA encoding KCNQ2 in 100 mM K+. e Mean current–voltage relationship for KCNQ2 in 100 mM K+ (black), Cs+ (blue), Rb+ (red), and Na+ (magenta), in the absence or presence of GABA (100 µM) as indicated; n = 5. f Estimated mean ion permeability relative to that of K+ versus ionic radius (Pauling) through KCNQ2 in the absence (black) and presence (purple) of GABA (100 µM); n = 5. g Representative traces from recordings of oocytes injected with cRNA encoding KCNQ3 in 100 mM K+. h Mean current–voltage relationship for KCNQ3* in 100 mM K+ (black), Cs+ (blue), Rb+ (red) and Na+ (magenta), in the absence or presence of GABA (100 µM) as indicated; n = 4. i Estimated mean ion permeability relative to that of K+ versus ionic radius (Pauling) for Na+, Rb+, and Cs+ through KCNQ3* in the absence (black) and presence (purple) of GABA (100 µM); n = 4. *p < 0.05. j Representative traces from recordings of oocytes injected with cRNA encoding KCNQ4 in 100 mM K+. k Mean current–voltage relationship for KCNQ4 in 100 mM K+ (black), Cs+ (blue), Rb+ (red), and Na+ (magenta), in the absence or presence of GABA (100 µM) as indicated; n = 4. l Estimated mean ion permeability relative to that of K+ versus ionic radius (Pauling) through KCNQ4 in the absence (black) and presence (purple) of GABA (100 µM); n = 4.
	Fig. 4. SMIT1–KCNQ complexes KCNQ-isoform dependently distinguish between GABA and related metabolites.All error bars indicate SEM. a Mean traces showing currents generated by KCNQ3* alone or with SMIT1 (n = 8). b Mean peak currents for KCNQ3* alone (black) or with SMIT1 (red) quantified from traces as in (a) (n = 8). c Mean peak tail currents for KCNQ3* alone (black) or with SMIT1 (red) quantified from traces as in (a) (n = 8). d Mean G/Gmax relationship for KCNQ3* alone (black) or with SMIT1 (red) quantified from traces as in (a) (n = 8). e Effects of SMIT1 on KCNQ3* activation rate quantified as a single exponential fit, time constant = τact (n = 8). p < 0.05. f Mean EM of unclamped oocytes expressing KCNQ3 alone (black) or with SMIT1 (red) (n = 8). p < 0.05. g Representative traces from KCNQ2 (Q2) in 100 mM K+ (voltage protocol inset). h Mean current–voltage relationship for Q2 in 100 mM K+ (black), Cs+ (blue), Rb+ (red), and Na+ (magenta), in the absence or presence of SMIT1 as indicated; quantified from traces as in (g); n = 5. i Estimated mean permeability relative to that of K+ versus ionic radius (Pauling) through Q2 in the absence (black) and presence (blue) of SMIT1; n = 5. p < 0.05; *p < 0.01. j Representative traces from KCNQ3 (Q3) in 100 mM K+ (voltage protocol as in (g)). k Mean current–voltage relationship for Q3 in 100 mM K+ (black), Cs+ (blue), Rb+ (red), and Na+ (magenta), with/without SMIT1 as indicated; quantified from traces as in (j); n = 4. l Estimated mean permeability relative to that of K+ versus ionic radius (Pauling) through Q3* with (red) or without (black) SMIT1; n = 4. *p = 0.006. m [3H]Myo-inositol uptake (30 min) for oocytes expressing KCNQ2 and SMIT1 in the absence or presence of neurotransmitters/metabolites indicated (100 µM) ([3H]Myo-inositol n = 10; GABA n = 16; BHB n = 17; GABOB n = 13). n [3H]Myo-inositol uptake (30 min) for oocytes expressing KCNQ3 and SMIT1 with/without neurotransmitters/metabolites indicated (100 µM) ([3H]Myo-inositol n = 17; GABA n = 10; BHB n = 8; GABOB n = 16).
	Fig. 5. KCNQ2/3 channels require an arginine at the foot of the voltage sensor for communicating GABA binding to SMIT1.All error bars indicate SEM. a Topology of KCNQ3 (two of four subunits shown) showing positions of W265 (red) and R242 (yellow) and segment numbering. VSD voltage-sensing domain. b Structural model of KCNQ3 showing positions of W265 and R242. Color coding as in (a). c Mean traces showing currents generated by KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) (n = 8) alone or with SMIT1 (n = 7) in Xenopus oocytes. d Mean peak currents for KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) alone (black; n = 8) or with SMIT1 (red; n = 7) quantified from traces as in (c). e Mean peak tail currents for KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) alone (black; n = 8) or with SMIT1 (red; n = 7) quantified from traces as in (c). f Mean G/Gmax relationship for KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) alone (black; n = 8) or with SMIT1 (red; n = 7) quantified from traces as in (c). g Mean EM of unclamped Xenopus oocytes expressing KCNQ2–W236L/KCNQ3–W265L (Q2WL/Q3WL) alone (black) (n = 8) or with SMIT1 (red) (n = 7). **p < 0.01. h Mean traces showing currents generated by KCNQ2–R213A/KCNQ3–R242A (Q2RA/Q3RA) alone (n = 14) or with SMIT1 (n = 16) in Xenopus oocytes. i Mean peak prepulse and peak tail currents for KCNQ2–R213A/KCNQ3–R242A (Q2RA/Q3RA) alone (black; n = 14) or with SMIT1 (red; n = 16) quantified from traces as in panel (h). j Mean EM of unclamped Xenopus oocytes expressing KCNQ2–R213A/KCNQ3–R242A (Q2RA/Q3RA) alone (black) (n = 14) or with SMIT1 (red) (n = 16). k [3H]Myo-inositol uptake (30 min) for Xenopus oocytes expressing subunits indicated in the absence or presence of GABA (100 µM) as indicated (SMIT1 alone, n = 19; Q2RA/Q3RA-S1, n = 29; Q2RA/Q3RA-S1 + GABA, n = 32).
	Fig. 6. Model of signaling in KCNQ2/KCNQ3–SMIT1 complexes.Schematic of KCNQ2/3–SMIT1 interaction showing functional effects of physical interaction and modulation by small molecules. DAG diacylglycerol, G6P glucose-6-phosphate, IMPase inositol monophosphatase, Ino-1 inositol-1 phosphate synthase, IP inositol phosphate, IPP inositol polyphosphatase 1-phosphatase, MI myo-inositol, PI4K phosphatidylinositol 4-kinase, PI5K phosphatidylinositol-5 kinase, PLC phospholipase C.

References [+] :

Abbott, KCNQ1, KCNE2, and Na+-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability. 2014, Pubmed, Xenbase