Fig 2: Baclofen has no direct effect on SLO2.1 channels. A, Whole-cell currents of SLO2.1 channels expressed in Xenopus oocytes before (control) and after the addition of 10 µM baclofen. The currents were evoked by voltage pulses from –100 to 40 mV in 10-mV steps at a holding potential of –80 mV. B, Representative I-V plot of currents from A shows that baclofen has no direct effect on SLO2.1 currents. Plot in C summarizes the current measurements at 40 mV before (control) and after baclofen treatment. Current values were normalized by control. Mean value (black horizontal line) and SD error bar are shown. There is no significant change in current amplitude after baclofen treatment (n = 5, p > 0.05).

Fig 3: Proposed model by which the NALCN/SLO2.1 complex regulates myometrial excitabilityDuring the quiescent state, progesterone binding to the progesterone receptor (PRA/B) increases NALCN expression and activity (Amazu et al., 2020). Sodium current through NALCN activates SLO2.1 channels, increasing K+ efflux to maintain the cell in a hyperpolarized state. As a result, voltage-dependent Ca2+ channels (VDCCs) are closed, and uterine contractions do not occur. In the contractile state, estrogen acting on ERa inhibits NALCN expression (Amazu et al., 2020), leading to decreased SLO2.1 activity. The reduced K+ efflux depolarizes the membrane, leading to VDCC activation, an increase in intracellular Ca2+, and uterine contractility. At labor, oxytocin (OXT) binds to the oxytocin receptor (OTR), leading to activation of phospholipase C (PLC), production of phosphatidyl inositol 4,5-bisphosphate (PIP2), and production of inositol triphosphate (IP3). IP3 activates the release of Ca2+ from intracellular stores, and PIP2 activates protein kinase C (PKC), which inhibits SLO2.1 (Ferreira et al., 2019). This SLO2.1 inhibition further depolarizes the membrane, thus opening more VDCCs, increasing intracellular Ca2+, and further activating myosin to cause muscle contraction. Figure designed and created by Anthony Bartley and Chrystie Tyler.

Fig 4: Activation of SLO2.1 by the NALCN-dependent Na+ leak hyperpolarizes the membrane potential(A) Experimental schemes and representative images of relative shifts of DiSC3(5) fluorescence induced by 80 mM sodium, 80 mM choline, 80 mM lithium, 10 µM gadolinium (Gd3+), and 50 µM CP96345 (NALCN Inhibitor) in hTERT-HM cells.(B) Quantification of shifts in cells by sodium (n = 26), choline (n = 15), lithium (n = 6), and sodium plus Gd3+(n = 5) or NALCN inhibitor (n = 6) normalized to changes in fluorescence in the presence of valinomycin. Data are presented as mean and standard deviation. **p < 0.01, ***p < 0.001 by unpaired t test with Mann-Whitney corrections.See Figures S2–S4.

Fig 5: The GABA-B receptor (GABAB-R1) colocalizes with sodium-activated potassium channels (SLO2.1) and voltage-sensitive sodium channels (NaV1.6). Rat olfactory bulb primary neurons were immunostained with pairwise combinations of primary antibodies (indicated at top left) raised from different species, followed by staining with fluorophore-coupled secondary antibodies. Panels c, f, and i are merged images, demonstrating that both proteins are present in the same cell. As with other channels and receptors, there is likely an intracellular pool as well a cell-surface pool, consistent with previous results (Panzanelli et al., 2004). Control samples incubated without any primary antibody show negligible staining (panels j, k, and l). The last column shows additional examples of immunostaining for SLO2.1 or NaV 1.6 in the absence or presence of the antigenic peptide (panels m, n, o, and p); the immunostaining is dramatically reduced in the presence of the peptide. Scale bar in p: 5 µm.