KCNQ2-5 channels contain GABA binding sites

Focusing on chemical properties required for anticonvulsant action of retigabine and related compounds on KCNQ2/3, including negative electrostatic surface potential localized close to a carbonyl oxygen13, we identified GABA as a candidate. GABA, the primary inhibitory neurotransmitter, bears strong negative electrostatic surface potential near its carbonyl oxygen, in contrast to the chemically related excitatory neurotransmitter, glutamate (Fig. 1d). Strikingly, in silico docking predicted binding of GABA to KCNQ3-W265, similar to retigabine and ML-213 (Fig. 1e). GABA does not readily cross the plasma membrane, but W265 lies in a crevice apparently accessible from the extracellular side (Fig. 1f); this portion of S5 also appeared accessible in the related Kv1.2–Kv2.1 “paddle-chimera” structure, which includes lipids14 (Fig. 1g). Accordingly, Xenopus oocyte expressed homomeric wild-type human KCNQ2–5-bound extracellularly applied 3H-GABA, while KCNQ1, which lacks W265, did not (Fig. 1h; Supplementary Fig. 1). Further indicative of the specificity of 3H-GABA binding to KCNQ2–5, non-injected oocytes bound minimal 3H-GABA (Supplementary Fig. 1) and 3H-GABA binding to homomeric wild-type human KCNQ3 was outcompeted by co-incubation with cold (unlabeled) GABA (1 mM) (Fig. 1i). Finally, results of saturation binding experiments conducted with oocyte-expressed homomeric wild-type human KCNQ3 using 10 different concentrations of 3H-GABA were consistent with binding to a specific site in KCNQ3 with a K d of 126 nM and a B max of 27 fmol per oocyte (Fig. 1j; see Supplementary Fig. 1d for an expanded view of the low [GABA] portion of the curve). The B max value was similar to results obtained from previous intact oocyte radioligand binding studies of other heterologously expressed membrane proteins15.

GABA activates KCNQ3 and KCNQ5

Using oocyte expression and two-electrode voltage clamp (TEVC), we found that GABA activates KCNQ3* (an expression-optimized KCNQ3-A315T mutant that ensures robust currents16) and KCNQ5, especially at subthreshold potentials. In contrast, the activity of KCNQ1, 2 and 4 was relatively GABA-insensitive (Fig. 2a; Supplementary Figures 2–6; Supplementary Tables 1–5). Thus, the KCNQ3-W265 equivalent is important for GABA binding (Fig. 1) but not sufficient for KCNQ activation by GABA (Fig. 2a). The KCNQ3* GABA EC 50 was 1.0 ± 0.1 µM at −60 mV (Supplementary Table 9), a higher sensitivity than that previously reported for retigabine (11.6 µM) or ML-213 (3.6 µM)13. This was a lower potency than the K d we observed for GABA binding to wild-type KCNQ3 (126 nM; Fig. 1j); but see GABA effects on KCNQ2/3-dependent membrane hyperpolarization, below (Fig. 2h). GABA (10 µM) shifted the V 0.5 of activation of KCNQ3* by −8 mV, but this understates the efficacy of GABA in the activation of KCNQ3*, because there is also a general increase in channel activity across the voltage range from −60 mV to +20 mV (Fig. 2a, b; Supplementary Fig. 4). The KCNQ5 GABA EC 50 was 0.06 ± 0.01 µM at −60 mV, a 17-fold higher GABA sensitivity than for KCNQ3* (Supplementary Table 9), but the efficacy of GABA with respect to channel activation was more than threefold greater for KCNQ3* compared to KCNQ5 (Fig. 2a, b). GABA (10 µM) shifted the KCNQ5 V 0.5 of activation by −15 mV (Supplementary Figure 6; Supplementary Table 5).

Fig. 2 KCNQ3 and KCNQ5 are activated by GABA in Xenopus oocytes. All error bars in figure indicate SEM. a Mean tail current versus prepulse voltage relationships recorded by TEVC in Xenopus laevis oocytes expressing homomeric KCNQ1–5 channels in the absence (black) and presence (purple) of GABA (n = 4–8). Voltage protocol as in c. b Mean GABA dose response at −60 mV for KCNQ1–5, quantified from data as in a (n = 5–8). c TEVC recordings using a current-voltage family protocol (upper left inset) in oocytes expressing KCNQ2/3 channels in the absence (black) and presence (purple) of GABA (10 µM). Dashed line indicates zero current level in this and all following current traces. d Averaged current traces at −60 mV for KCNQ2/3 channels in the absence (black) and presence (purple) of GABA (10 µM) (n = 10). Voltage protocol upper inset. e Left, mean tail current; right, mean normalized tail current (G/Gmax), both measured at arrow in voltage protocol (center) from traces as in c, with versus without GABA (10 µM) (n = 10). f Mean voltage dependence of KCNQ2/3 current fold-increase by GABA (10 µM), plotted from traces as in c (n = 10). g Mean dose response of KCNQ2/3 channels at −60 mV for GABA (calculated EC 50 = 0.85 µM; n = 10) and glutamate (no effect; n = 5). h GABA hyperpolarizes resting membrane potential (E M ) of unclamped oocytes expressing KCNQ2/3. Left, effects of 10 µM GABA; right, dose response; n = 10, ****P < 0.0001. i Exemplar −60 mV KCNQ2/3 current before (left, black), during wash-in of GABA (purple) and after wash-out (right, black). j Exemplar −60 mV KCNQ2/3 current before (left, black), during wash-in of GABA (purple), wash-in of XE991 (red), and after wash-out (right, black). Membrane potential was clamped at −60 mV except for a 2-min pulse to +60 mV during the early phase of GABA wash-in. k Mean activation (left) and deactivation (right) rates for KCNQ2/3 before (control) and after wash-in of GABA (n = 10); **P < 0.01; ***P = 0.0007. Activation rate was quantified using voltage protocol as in c. Deactivation rate was quantified using voltage protocol shown above Full size image

GABA activation of KCNQ2/3 channels

We next focused on heteromeric KCNQ2/3 channels, the primary molecular correlate of neuronal M-current4. GABA activated KCNQ2/3 with increasing efficacy at more negative membrane potentials, a property important for subduing neuronal activity; thus GABA (10 µM) negative-shifted the V 0.5 of KCNQ2/3 activation by −14 mV (Fig. 2c–e; Supplementary Fig. 7; Supplementary Table 6). At −60 mV, 10 µM GABA increased KCNQ2/3 current fourfold (Fig. 2f). The EC 50 for KCNQ2/3 activation was 0.85 ± 0.1 µM GABA at −60 mV (Supplementary Table 9), while the excitatory neurotransmitter glutamate had no effect on KCNQ2/3 even at 10 mM (Fig. 2g; Supplementary Fig. 8; Supplementary Table 7). Reflecting its greater efficacy at more negative membrane potentials and the efficiency of KCNQ channels in setting membrane potential (E M ), GABA exerted potent effects on KCNQ2/3-dependent membrane hyperpolarization (EC 50 , 120 nM) (Fig. 2h). Canonical pentameric GABA A receptors, which are triggered to open when GABA binds to an extracellularly located, inter-subunit GABA-binding site, participate in either phasic or tonic inhibition, depending on their location and subunit composition17, 18. Tonic extracellular GABA concentration in mammalian brain is calculated to be ~160 nM19, 20, and transient peak GABA concentrations of several millimolar occur in the synaptic cleft21. Canonical α x β 3 γ 2 GABA A Rs exhibit GABA EC 50 values of 1.0–157 µM22, 23. Thus, KCNQ2/3 channel GABA affinity compares to that of the most sensitive α x β 3 γ 2 GABA A Rs. GABA begins to activate KCNQ2/3 immediately on wash-in; the current augmentation takes ~3 min to plateau, and persists during wash-out (Fig. 2i). Time to onset of functional effects upon wash-in did not appear voltage-dependent, as it was not altered by switching to +60 mV during wash-in; the GABA-augmented current was readily inhibited by washing in the KCNQ channel inhibitor, XE991 (50 µM) (Fig. 2j). GABA accelerated KCNQ2/3 activation and slowed deactivation, suggesting GABA stabilizes the KCNQ2/3 open state (Fig. 2k; Supplementary Fig. 9; Supplementary Table 8).

GABA activates KCNQ2/3 channels by binding to KCNQ3-W265

Our radioligand binding data (Fig. 1h–j) demonstrate direct binding of GABA to KCNQ2–5, but we nevertheless tested for other possibilities and artifacts. At 100 µM, GABA neither induced current in, nor shifted the resting membrane potential of, water-injected oocytes, demonstrating our observations did not arise from an endogenous ion channel (Fig. 3a), neither did increasing GABA to 1 mM produce any noticeable effects (Supplementary Fig. 10). Neither did GABA, even at 1 mM, affect activity of KCNA1 (Kv1.1), a Kv channel from a different subfamily (Fig. 3b; Supplementary Fig. 11; Supplementary Table 10). GABA B receptors are metabotropic receptors that can activate some potassium channels via pertussis-sensitive G proteins24. In previous studies, it was concluded that Xenopus oocytes do not express endogenous GABA B receptors25, which can activate some potassium channel subtypes (not reported for KCNQs), but we nevertheless tested for their possible role in the observed effects, using four approaches. These studies ruled out a role for GABA B receptors in KCNQ activation by GABA, as follows. First, GABA activated KCNQ2/3 even after overnight treatment of oocytes with pertussis toxin, which inhibits G proteins associated with GABA B activation of some potassium channels (Fig. 3c; Supplementary Fig. 12; Supplementary Table 11). Second, KCNQ2/3 channels were insensitive to baclofen (100 µM), a GABA analog that acts as a GABA B R agonist26 (Fig. 3d; Supplementary Fig. 13; Supplementary Table 12). Third, GABA B receptor antagonist saclofen neither altered KCNQ2/3 currents alone, nor did it diminish GABA activation of KCNQ2/3 (Fig. 3e; Supplementary Figs. 14, 15; Supplementary Tables 13, 14). Fourth, the potent GABA B receptor antagonist CGP55845 neither altered KCNQ2/3 currents alone, nor did it diminish GABA activation of KCNQ2/3 (Fig. 3f; Supplementary Figs. 16, 17; Supplementary Tables 15, 16).

Fig. 3 GABA directly opens KCNQ2/3 channels. All error bars in figure indicate SEM. a TEVC of water-injected Xenopus laevis oocytes showing no effect of GABA (100 µM) on endogenous currents or (lower right) resting membrane potential (E M ) (n = 4–5). b TEVC of Xenopus laevis oocytes expressing KCNA1 (Kv1.1) showing no effect of GABA (1 mM) on peak current (left) or normalized tail current (right) (n = 4). c–f GABA effects on KCNQ2/3 in oocytes do not require GABA B receptor activity. Mean KCNQ2/3 tail currents in oocytes showing GABA activates KCNQ2/3 in the presence of pertussis toxin (2 µg/ml), saclofen (100 µM), or CGP55845 (100 µM) and that neither baclofen (100 µM), saclofen, nor CGP55845 alter KCNQ2/3 current independently (n = 5–7). g Upper, tail current; lower, normalized conductance; showing mean GABA response of oocyte-expressed KCNQ2/KCNQ3-W265L (n = 5). h Upper, tail current; lower, normalized conductance; showing mean GABA response of oocyte-expressed KCNQ2-W236L/KCNQ3-W265 (Q2/Q3-WL/WL) (n = 5). i Mean current fold-changes (upper) and dose responses (lower) for channels as indicated; KCNQ2/KCNQ3 results (purple line) and KCNQ3* results (red line) from Fig. 2 shown for comparison; n = 5–10. j Mean 3H-GABA binding for KCNQ2/3 versus KCNQ2-W236L/KCNQ3-W265L channels expressed in Xenopus oocytes; n = 73 (Q2/Q3), 29 (KCNQ2-W236L/KCNQ3-W265L) in two to four batches of oocytes; ****P < 0.0001 Full size image

Mutagenesis studies further cemented the conclusion that GABA directly modulates KCNQ2/3 channels. Reflecting our in silico predictions, 3H-GABA binding results (Fig. 1e–j), and KCNQ2 GABA-insensitivity (Fig. 2a), a mutant KCNQ2/3 channel complex in which the essential KCNQ3 tryptophan was replaced with leucine (KCNQ2/KCNQ3-W265L) was GABA-insensitive (up to 10 mM), as was double tryptophan-mutant heteromer KCNQ2-W236L/KCNQ3-W265L (Fig. 3g–i; Supplementary Figs. 18, 19; Supplementary Table 17), which also showed impaired 3H-GABA binding (Fig. 3j).

GABA activates endogenous neuronal M-channels

Switching to mammalian cells, KCNQ2/3 channels heterologously expressed in Chinese hamster ovary (CHO) cells were likewise activated by GABA, demonstrating this was not an oocyte-specific phenomenon (Fig. 4a, b). GABA (100 µM) shifted the midpoint voltage dependence of KCNQ2/3 activation by −14.6 mV (from −2.9 to −17.5 mV, Supplementary Table 18), similar to the shift we observed in oocytes (−14 mV; Fig. 2c and Supplementary Table 6).

Fig. 4 GABA activates KCNQ2/3 in CHO cells and native M-current. All error bars in figure indicate SEM. a Exemplar current traces from CHO cells transfected to express KCNQ2/3 channels, recorded using whole-cell patch clamp, showing effects of 100 µM GABA (n = 6). b Mean normalized tail current from recordings as in a (n = 6). c Representative micrographs (left) and whole-cell currents (right) from undifferentiated (upper) versus nerve growth factor (NGF)-differentiated (lower) PC12 cells. Scale bars, 10 µm. d Upper, representative tail currents (using voltage protocol as in c); lower, mean normalized tail currents, recorded from NGF-differentiated PC12 cells bathed in extracellular solution alone (control) or with picrotoxin (100 µM) and CGP55845 (10 µM) to block GABA A and GABA B receptors, respectively, alone (black square) or in combination with GABA (100 µM; purple circle) or XE991 (10 µM; open red square); n = 7). e Representative tail currents (using voltage protocol on left) recorded from mouse DRG neurons bathed in extracellular solution containing picrotoxin (100 µM) and CGP55845 (10 µM), alone (black) or in combination with GABA (100 µM; purple) or GABA + XE991 (10 µM; red). f Mean current at −60 mV divided by current at −20 mV in the same DRG neuron, using the protocol as in e, in the absence (control) or presence of GABA (n = 7); **P = 0.005. g Mean GABA-dependent increase in current at −20 mV versus at −60 mV in DRG neurons, using the protocol as in e (n = 7); P = 0.07 between groups. h Mean effect of GABA versus GABA + XE991 on the magnitude of the deactivating current at −60 mV, using the protocol as in e (n = 5); P = 0.1 between groups Full size image

We next tested the effected of GABA on native M-current in PC12 cells, a rat pheochromocytoma cell line of neural crest origin that retains many of the properties of dopaminergic neurons and has previously been used to study M-current27, 28. Culturing PC12 cells for 4–7 days with NGF resulted in extensive neurite outgrowth and increased expression of native voltage-gated potassium currents (Fig. 4c), as previously reported27, 28. We found that 8/10 PC12 cells studied expressed potassium current sensitive to the KCNQ channel blocker XE991 (10 µM). Importantly, in seven of these eight cells, GABA (100 µM) negative-shifted the voltage dependence of the PC12 cell tail current activation, in the presence of picrotoxin and CGP55845 to block native canonical GABA A and GABA B receptors, respectively. The mean shift of −13.5 mV in the midpoint of voltage-dependent activation in PC12 cells in response to 100 µM GABA, in the presence of canonical GABAR blockers (Supplementary Table 19), was similar to that for KCNQ2/3 channels expressed in oocytes and in CHO cells (−14 mV) with 100 µM GABA application. Application of KCNQ channel blocker XE991 (10 µM) removed the GABA-dependent negative-shift in the PC12 cell tail currents (Fig. 4d).

We also recorded the effects of GABA on dorsal root ganglion (DRG) neurons isolated from adult mice, using perforated patch-clamp whole-cell recordings. Cells were held at −20 mV to inactivate other Kv channels and then pulsed to −60 mV, after wash-in of inhibitors of canonical GABA A and GABA B receptors (100–300 µM picrotoxin; 10 µM CGP55845, respectively), first in the absence, and then in the presence, of GABA (100 µM). XE991 (10 µM) was subsequently washed in, in the presence of the other drugs, to inhibit M-current. From a holding potential of −20 mV, pulses to −60 mV produced a partial deactivation and then stabilization characteristic of M-current; current at −60 mV was increased by GABA and this current was decreased by XE991 (Fig. 4e). GABA (100 µM) increased the relative proportion of −60 mV current to that of −20 mV current, consistent with greater GABA efficacy at more negative voltages (Fig. 4f). Accordingly, the mean GABA-dependent current increase was threefold at −60 mV, but there was no mean change in current at −20 mV (Fig. 4g). We also quantified the deactivating portion of the −60 mV current in isolation. GABA decreased this deactivating current by ~25%, consistent with fewer channels closing upon the transition from −20 mV to −60 mV, in the presence of GABA. XE991 applied in combination with GABA inhibited this deactivating current >50%, consistent with the majority of the current being passed by KCNQ channels (Fig. 4h). Thus, GABA activation of M-channels is direct, independent of the expression system, and occurs with both exogenously and endogenously expressed channels.

GABA overcomes muscarinic inhibition of KCNQ2/3 channels

Muscarinic inhibition of KCNQ channels is achieved by disruption of KCNQ regulation by phosphatidylinositol 4,5-bisphosphate (PIP 2 ). PIP 2 facilitates KCNQ channel opening, thus hyperpolarizing KCNQ channel voltage-dependent activation, enabling opening at more negative membrane potentials. Muscarinic acetylcholine receptor (mAChR) activation disrupts this, by hydrolyzing PIP 2 and/or by reducing PIP 2 sensitivity, thus positive-shifting KCNQ channel voltage dependence of activation, inhibiting activity particularly at negative potentials29. The lack of GABA responsiveness of KCNQ1, 2, and 4 (Fig. 2a) effectively ruled out the possibility that endogenous mAChRs/changes in PIP 2 were mediating GABA effects on KCNQ3. However, we further tested this and also asked whether GABA could overcome muscarinic inhibition of KCNQ2/3. For these experiments, we reverted to the Xenopus oocyte expression system. As expected, application of acetylcholine (ACh) to activate endogenous oocyte mAChRs inhibited KCNQ2/3 activity, especially at negative voltages; maximal effect occurred at 1 µM ACh and higher (Fig. 5a, b; Supplementary Fig. 20; Supplementary Table 20). For comparison, application of a 100-fold greater concentration of dopamine (100 µM) had no effect on KCNQ2/3 current (Fig. 5c; Supplementary Fig. 21; Supplementary Table 21). ACh inhibition of KCNQ2/3 was prevented by the mAChR antagonist atropine, indicating that ACh inhibited KCNQ2/3 solely by the canonical, indirect pathway requiring mAChR activation (Fig. 5b, d; Supplementary Fig. 22). Atropine itself had no direct effect on KCNQ2/3 activity (Fig. 5b, e; Supplementary Fig. 23; Supplementary Table 22). Additionally, atropine did not inhibit GABA activation of KCNQ2/3 (Fig. 5f; Supplementary Fig. 24; Supplementary Table 23), ruling out an indirect, mAChR-dependent mechanism for GABA activation of KCNQ2/3, as expected from the GABA-insensitivity of other PIP 2 -sensitive KCNQs (Fig. 2a).

Fig. 5 GABA activation is independent of and overrides muscarinic inhibition of KCNQ2/3 in Xenopus oocytes. All error bars in figure indicate SEM. a Mean dose response for acetylcholine (ACh) on KCNQ2/3 activity in oocytes (n = 5). b Mean effects versus voltage for ACh or atropine alone or in combination on KCNQ2/3 activity in oocytes (n = 5). c Lack of effects of dopamine (100 µM) on KCNQ2/3 mean tail currents in oocytes (n = 6). d Atropine blocks KCNQ2/3 inhibition by ACh (measured using tail currents in oocytes; n = 5). e Lack of effects of atropine (100 µM) on KCNQ2/3 mean tail currents in oocytes (n = 5). f Atropine does not prevent GABA activation of KCNQ2/3 measured via tail currents in oocytes (n = 5). Left, mean tail currents, right, current fold-change versus voltage for GABA + atropine versus no drugs. g GABA prevents inhibition of KCNQ2/3 by ACh, measured via tail currents in oocytes (n = 5). Left, mean tail currents, right, current fold-change versus voltage for GABA + ACh versus no drugs. h GABA (10 µM) effects on KCNQ2/3 in oocytes are not prevented by partial depletion/inhibition of synthesis of PIP 2 using wortmannin (30 µM for 3 h) (n = 5). Left, mean tail current; right, GABA dose response at −60 mV; (n = 5). i GABA (100 µM) has no effect on KCNQ1/KCNE1. Left, averaged traces; right, mean tail currents (n = 6). j GABA (100 µM) does not alter resting membrane potential (E M ) of unclamped oocytes expressing KCNQ1-KCNE1 (n = 6) Full size image

Importantly, GABA overcame ACh-dependent KCNQ2/3 inhibition (Fig. 5g; Supplementary Fig. 25; Supplementary Table 24), indicating that GABA can override the canonical muscarinic inhibition of KCNQ2/3 for which the M-current is named. To further test this conclusion, we pre-incubated KCNQ2/3-expressing oocytes with the phosphoinositide 3/4-kinase inhibitor, wortmannin (30 µM for 3 h), to prevent PIP 2 synthesis and partially deplete intracellular PIP 2 . Under these conditions, KCNQ2/3 was still robustly activated by GABA, which now (at 10 µM) shifted the V 0.5 of KCNQ2/3 activation by −24 mV (Fig. 5h; Supplementary Fig. 26; Supplementary Table 25). Thus, GABA activation of KCNQ2/3 can overcome the inhibitory effects of mAChR activation or direct PIP 2 depletion/prevention of PIP 2 synthesis. As a final control for the independence of GABA from mAChR regulation, we examined effects of GABA on KCNQ1-KCNE1, a heteromeric channel with 100-fold higher PIP 2 sensitivity than homomeric KCNQ130. We found no effect of GABA on KCNQ1-KCNE1 activity, nor on the resting membrane potential of KCNQ1-KCNE1-expressing oocytes (Fig. 5i, j; Supplementary Fig. 27; Supplementary Table 26). Thus, KCNQ activation by GABA does not involve mAChRs or PIP 2 , and indeed can overcome these factors in KCNQ2/3 channels.

GABA analogs compete for KCNQ3-W265 binding

GABA coexists in the CNS with various analogs and metabolites, two of which, β-hydroxybutyrate (BHB) and γ-amino-β-hydroxybutyrate (GABOB), we discovered to also activate KCNQ2/3. GABOB had a higher affinity (EC 50 of 0.12 µM), but was a lower efficacy partial agonist, for KCNQ2/3 compared to BHB (EC 50 of 0.94 µM) and to GABA (EC 50 of 0.85 µM). In contrast, the structurally related straight-chain alkyl carboxylic acid, valeric acid, was inactive up to 5 mM (Fig. 6a, b; Supplementary Figs. 28–30; Supplementary Tables 27–29). The high sensitivity of KCNQ2/3 for BHB is in contrast with that of canonical GABA A receptors, which are BHB-insensitive up to 5 mM BHB31. In silico docking studies suggested that, like GABA, BHB, and GABOB bind to KCNQ3-W265 (Fig. 6c). Accordingly, GABOB, a higher-affinity partial agonist compared to GABA, diminished the KCNQ2/3-activating efficacy of GABA (Fig. 6d; Supplementary Fig. 31; Supplementary Table 30). In contrast and as expected, glutamate, which we found to be inactive on KCNQ2/3 (Fig. 2g), did not alter GABA activation of KCNQ2/3 (Fig. 6e; Supplementary Fig. 32; Supplementary Table 31). Finally, we found that GABOB reduces the efficacy of retigabine, an anticonvulsant that also activates KCNQ2/3 by binding to the W265 equivalent (Fig. 6f).