Lithium inhibition of NMDA-elicited currents
Cortical neurons in cultures express a variety of NCXs including NCX1-3 and NCKX isoforms [14]. Extracellular Li+ represents a tool to cause the substrate inhibition of Na+-dependent Ca2+ extrusion by all sodium-calcium exchanger subtypes. The stepwise proportional substitution of Li+ for Na+ in the bathing solution was used to obtain the dose-inhibition curve of NMDA-evoked currents for Li+. With this particular aim the NMDA-activated currents were measured at 0, 21, 42, 70, 112 and 140 mM Li+ in the bathing solution in the same experiment, where 140 mM Li+ corresponded to 100% substitution of Li+ for Na+. An application of Li+-containing solutions without agonists always preceded the application of the corresponding solution with NMDA. An increase of Li+ concentrations in the external solution caused a decrease of NMDA-activated currents at the steady state (Fig. 1a). The control NMDA-evoked currents, measured at the steady state in the bathing solutions (140 mM Na+) had the amplitude of 440.4 ± 71.9 pA (n = 10), that was significantly (p < 0.001, Student’s two-tailed t test) larger compared to the corresponding value of 111.4 ± 29.1 pA (n = 10) measured at 140 mM Li+ in the external solution. Dose-inhibition curves obtained from experiments were well fitted by Hill equation with IC50 of 46 ± 21 mM (Fig. 1b). Previously, we demonstrated that the inhibition of NMDA-activated currents by Li+ is Ca2+-dependent, because it could not be observed in the nominal absence of Ca2+ in the external solution [11]. Since Li+ does not directly affect the NMDAR conductance and activation kinetics, as a substrate inhibitor of NCXs it could sufficiently decrease the efficacy of Ca2+ extrusion from neurons due to breaking ion transport by NCXs. The decrease of NMDAR current by Li+ suggests that NCX contributes to the regulation of free Ca2+ concentration close to the inner membrane surface and the Ca2+-dependent desensitization of NMDARs. This requires some functional interaction between NCXs and NMDARs that could occur if these molecules are located closely and interact within lipid nanoclasters or rafts.
The extraction of cholesterol from the plasma membrane by MβCD [17] is a widely used conventional procedure to destroy lipid nanoclusters. The treatment of neurons with 1.5 mM MβCD for 5 min was undertaken to extract cholesterol from membrane lipid rafts to achieve spatial uncoupling of NMDARs and NCXs. This procedure did not significantly alter the current–voltage relationships (I/V), suggesting the lack of its effect on the input resistance of neurons (n = 5, Fig. 1a). After the cholesterol extraction the mean amplitude of NMDA-evoked currents at the steady state in the Na+-containing bathing solution was 136.8 ± 32.8 pA (n = 10), revealing its decrease in comparison to MβCD untreated neurons as control conditions (p < 0.007, Student’s two-tailed t test, Fig. 1a, b). The stepwise substitution of Li+ for Na+ in the external solution after the MβCD treatment further decreased the NMDA-evoked currents to the mean steady-state amplitude of 46.8 ± 15.3 pA (n = 10, p < 0.008, Student’s two-tailed t-test). The IC50 value for the Li+ inhibition of NMDA-activated currents after the MβCD treatment was 42 ± 20 mM (Fig. 1b, c) which did not differ significantly from the value obtained under the control conditions (on MβCD untreated neurons). It should be noted, however, that the degree of the NMDAR current inhibition in the Li+-containing bathing solution was less pronounced after the MβCD treatment than before this procedure and were 59 ± 4% (n = 10) and 77 ± 3% (n = 10) inhibition (p < 0.03, Student’s two-tailed t-test), respectively (Fig. 1d). Presumably, spatial uncoupling of NCXs and NMDARs limits the effect of the NCX inhibition on NMDAR currents. This could be the case, if NCXs maintain low intracellular free Ca2+ concentration in the close proximity of NMDARs, which prevents the development of Ca2+-dependent inactivation of NMDARs.
Calcium-dependent and -independent effects of cholesterol extraction on NMDARs
The interpretation of the above data that the cholesterol extraction may accelerate the Ca2+-dependent desensitization destroying membrane lipid rafts and NCX-NMDAR interplay becomes less evident in a view of the recent observation that cholesterol is important for the NMDAR functioning and its extraction provokes the ligand-dependent desensitization of NMDARs [17]. In order to distinguish between Ca2+-dependent and -independent mechanisms the effects of cholesterol extraction by MβCD on NMDA-activated currents were evaluated in the presence of 1 mM Ca2+ and in the nominal absence of Ca2+ in the bathing solution (Fig. 2a).
In the absence of Ca2+ in the external solution the ratio of amplitudes of NMDA-activated steady-state currents, recorded after and before 5 min MβCD treatment was 47 ± 6% (n = 6). The decrease of the steady-state amplitudes of NMDAR currents after the treatment is caused by the direct effect of the cholesterol extraction on NMDARs, because under these particular conditions the Ca2+-dependent desensitization was not observed (Fig. 2a). In the presence of 1 mM Ca2+ in the bathing solution, however, the Ca2+-dependent desensitization of NMDARs, measured as the ratio of the steady-state amplitudes of currents in the presence and absence of Ca2+ before and after the MβCD treatment was significantly greater when cholesterol was extracted (Fig. 2a, b), suggesting that this procedure enhanced the Ca2+-dependent NMDAR desensitization. In addition, we performed similar experiments on neurons patched with 1 mM BAPTA in the pipette solution. Under these particular conditions the Ca2+-dependent desensitization of NMDARs was not observed both in the presence and absence of Ca2+ in the external bathing solution (Fig. 2c). The direct effect of MβCD treatment on NMDARs was pronounced and the ratio values obtained in the presence and absence of external Ca2+ were similar (Fig. 2c, d). In 1 mM intrapipette BAPTA the steady-state NMDAR currents decreased after the extraction to about 10% of their amplitudes (Fig. 2d), whereas in experiments when the intracellular media was natural in terms of Ca2+ buffering the NMDAR currents decreased in a much lesser extent (about 47%, Fig. 2a).
Based on these experiments we may assume that in lipid rafts NCX weakens Ca2+-dependent desensitization of NMDARs by quick extrusion of local intracellular Ca2+ entering neurons via open NMDAR pores. It is likely, that the destruction of lipid rafts increases the distance between NCXs and NMDARs allowing intracellular Ca2+ accumulation close to the NMDAR intracellular domains which enhances their Ca2+-dependent desensitization. Pronounced Ca2+-dependent desensitization of NMDARs, however, should provide a feed back regulation to limit the cytoplasmic Ca2+ accumulation during the NMDA action on neurons.
NCX inhibition and NMDA-elicited cytoplasmic Ca2+ accumulation
To provide additional experimental support in favor of mechanisms suggested, the effects of NCX inhibition with 140 mM Li+ or KB-R7943 before and after the cholesterol extraction by MβCD (1.5 mM for 5 min) on intracellular Ca2+ responses to 2 min NMDA applications were studied. For quantitative comparison of effects we evaluated an integral of Ca2+-induced fluorescence, which has to be proportional to the Ca2+ entry through open NMDAR channels and, therefore, to the amplitudes of NMDA-activated currents. As in electrophysiological experiments, the Li+-containing bathing solution was applied alone and than with NMDA to equilibrate neurons and check pure Li+ effects for possible further data correction (Fig. 3a). When NMDA was applied in the Li+-containing bathing solution Ca2+ responses of neurons decreased to 54 ± 2% (overall 98 neurons, n = 3) of Ca2+ responses recorded in the Na+-containing bathing solution (p < 0.001, Student’s t test). This observation is consistent with the Li+ effect on NMDA-activated currents. After the MβCD treatment the Ca2+ responses to NMDA in the Na+-containing solution were 35 ± 9% (overall 98 neurons, n = 3) and in the Li+-containing solution were 36 ± 9% (overall 98 neurons, n = 3) of the Ca2+ responses, obtained before the treatment in the Na+-containing solution (Fig. 3a and b). Because these values were significantly smaller, than those obtained before the treatment in the Na+ solution (p < 0.0001, one-way paired ANOVA) and did not differ between each other (Bonferroni post hoc test) we conclude that the MβCD treatment abolished the effects of Li+ on NMDARs.
Thus, spatial uncoupling of NMDARs and NCXs resulted in the decrease of Ca2+ entry via NMDARs. Inhibition of NCX with Li+ after the cholesterol extraction was not able to decrease NMDAR mediated Ca2+ accumulation.
We further performed the Ca2+ imaging experiments in which KB-R7943 (10 µM) as a specific NCX inhibitor, was utilized instead of the Li+ solution. In the Na+-containing external solution combined applications of NMDA with KB-R7943 induced Ca2+ responses that corresponded to 59 ± 5% (overall 91 neurons, n = 3) of NMDA-elicited Ca2+ responses and differed from them significantly (p < 0.001, one-way paired ANOVA) (Fig. 3c). This observation is consistent with the KB-R7943 effect on NMDA-activated currents [11]. The MβCD treatment decreased the NMDA-elicited Ca2+ responses both in the absence and presence of KB-R7943 to 32 ± 8% and 24 ± 7% (overall 91 neurons, n = 3), respectively (Fig. 3d). These values are not significantly different (Bonferroni post hoc test) suggesting that the cholesterol extraction abolished the KB-R7943 effects on NMDA-activated currents. To validate that the effects of MβCD treatment are actually caused by the cholesterol loss from the plasma membrane, cholesterol-MβCD, as a cholesterol donor, was applied for 30 min after the effects of MβCD were achieved (Fig. 3c). Loading of cholesterol into the plasma membrane both increased the amplitudes of Ca2+-responses to NMDA and recovered the inhibitory effect of KBR (Fig. 3c, d).
Therefore, the effects of Li+ and KB-R7943 on NMDA-elicited Ca2+ responses of neurons coincide well suggesting that they both are realizing through the influence of NCX on the Ca2+-dependent desensitization of NMDARs.