Mg2+ block properties of triheteromeric GluN1-GluN2B-GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones

Abstract

Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg(2+) is an important regulator of NMDARs, and particularly the voltage dependence of Mg(2+) block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg(2+) block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1-GluN2B-GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage-dependent Mg(2+) block properties of triheteromeric NMDARs. In control conditions, external Mg(2+) significantly inhibits the whole cell NMDA-evoked current in a voltage-dependent manner with IC50 values of 20.9 μm, 53.3 μm and 173 μm at -90 mV, -70 mV and -50 mV, respectively. When the GluN2B-selective antagonist ifenprodil was applied, the Mg(2+) sensitivity of the residual NMDA-mediated currents (which is mainly carried by GluN1-GluN2B-GluN2D NMDARs) is reduced to IC50 values of 45.9 μm (-90 mV), 104 μm (-70 mV) and 276 μm (-50 mV), suggesting that triheteromeric GluN1-GluN2B-GluN2D NMDARs have less affinity for external Mg(2+) than GluN1-GluN2B receptors. In addition, fitting INMDA-V curves with a trapping Mg(2+) block model shows the triheteromeric GluN1-GluN2B-GluN2D NMDARs have weaker voltage-dependent Mg(2+) block (δ = 0.56) than GluN1-GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1-GluN2B-GluN2D rather than GluN1-GluN2D NMDARs are present. These data provide information relevant to Mg(2+) block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.

Figure 1. Experimental protocol and dopaminergic neurone identification

A, example whole cell voltage clamp traces recorded from postnatal day 7 dopaminergic neurone in substantia nigra pars compacta. The neurone was voltage clamped at −60 mV and inward NMDA-mediated currents were activated by bath application of 20 μm NMDA and 10 μm glycine. Three concentrations of Mg²⁺ (0 mm, 0.1 mm and 1 mm) were tested. Bars above the current trace show the time of application of indicated solutions. Vertical lines on the NMDA-mediated current trace are current responses evoked by voltage ramps from −100 mV and +40 mV. B, left, expanded voltage ramp current trace from a recording illustrated in (A). Each current trace was obtained by averaging four repeated trails. B, right, example I_NMDA–V relations obtained from (A), by subtracting the control current trace from currents recorded in the presence of NMDA with resultant I_NMDA values plotted versus membrane potential. C, example Differential Interference Contrast images of typical postnatal day 7 rat dopaminergic neurones. The scale represents 20 μm. D, example characteristic I_h current used for identification of dopaminergic neurones. White dashed line on the current trace shows single exponential fitting and fitting results are shown where (A) is predicted maximum I_h current and τ is the activation time constant.

Scheme 1. Sequential block model

Scheme 2. Trapping block model

Figure 2. External Mg²⁺ inhibits NMDARs in dopaminergic neurones in a concentration- and voltage-dependent manner

A, graph depicting the mean and standard error of whole cell I_NMDA measured at −60 mV in the absence and in the presence of external Mg²⁺. Open circles illustrate I_NMDA from individual experiments. B, example of I_NMDA–V relations obtained from a single neurone. C, averaged and normalized I_NMDA–V relations obtained from 26 neurones. B and C, Mg²⁺ concentrations indicate the added Mg²⁺ concentrations without taking background Mg²⁺ into account. D, concentration–inhibition curves for external Mg²⁺ inhibition of NMDARs in postnatal day 7 dopaminergic neurones. The contaminating Mg²⁺ concentration (27.9 μm) in the ‘Mg²⁺-free’ solution was estimated by fitting the Mg²⁺ blocking model, which is shown in Fig.4.

Figure 3. Residual whole cell NMDA currents recorded in the presence of 10 μm ifenprodil displayed weak voltage-dependent Mg²⁺ block

A, example whole cell NMDAR-mediated current trace obtained from a postnatal day 7 dopaminergic neurone at a holding potential of −60 mV. B, mean and standard error of whole cell I_NMDA obtained in the absence (Ctr) and in the presence of ifenprodil is shown with the values (open circles) of I_NMDA obtained from individual experiments. C, averaged (means ± s.e.m.; n = 16) and normalized I_NMDA–V relations recorded in the presence of 10 μm ifenprodil. D, Mg²⁺ concentration–inhibition curves for ifenprodil insensitive NMDARs. The calculated contaminating Mg²⁺ (27.9 μm) in acute slices was predicted using the Mg²⁺ blocking model shown in Fig.4.

Figure 4. Estimation of voltage-dependent parameters of Mg²⁺ block with the trapping block model

A and B, averaged and normalized I_NMDA–V relations obtained in the absence (A), and in the presence (B), of ifenprodil are shown fitted using the trapping block model to estimate the voltage-dependent Mg²⁺ block parameters of K_Mg (0 mV) and δ and the background Mg²⁺ concentration. C, sensitivity analysis 3D plot illustrates the sum of squares of the trapping block model fit to the data with varied K_Mg (0 mV) and δ. The red crosses indicate the 95% confidence intervals derived from curve fitting in the control and ifenprodil conditions.

Figure 5. Deactivation and desensitization kinetics of NMDARs in postnatal day 7 dopaminergic neurones

A, example concentration jump recordings obtained from an outside-out patch. NMDAR-mediated single channel current was evoked by a 1 ms pulse of 1 mm glutamate and 10 μm glycine. The expanded trace shows that both high-conductance and low-conductance NMDARs are present in this patch. Ba, open tip experiment used to estimate the onset of glutamate application and to optimize the best position of recording pipette. Bb, example macroscopic current from one individual patch. Its falling phase of the trace was well fitted with two exponential components. Bc, averaged macroscopic NMDAR-mediated current from six individual patches. Ca, example macroscopic NMDAR-mediated current obtained from an outside-out patch during a 4 s pulse of 1 mm glutamate and 10 μm glycine. The macroscopic current shows a significant desensitization following the activation of NMDARs. Cb and Cc, exemplary and averaged (n = 4 patches) macroscopic NMDAR-mediated currents in response to 4 s glutamate application.

Figure 6. Voltage-dependent Mg²⁺ block of NMDARs is independent of agonist concentration

A and B, showing the average and standard error values of normalized NMDA-mediated currents obtained at different voltages in the presence of contaminating background (A) or 100 μm of external Mg²⁺ (B) (for clarity, 300 μm and 1000 μm Mg²⁺ data are not shown). There is no significant difference in extent of Mg²⁺ block between NMDA-mediated current activated by 250 μm NMDA and 20 μm at four external Mg²⁺ concentrations (background, 100 μm, 300 μm and 1 mm of Mg²⁺), suggesting that the voltage-dependent Mg²⁺ block properties are independent of NMDA (agonist) concentration. Ifen., ifenprodil.

Figure 7. Comparison of Mg²⁺ sensitivity between residual NMDAR-mediated currents obtained in the presence of ifenprodil and normal NMDAR-mediated whole cell currents

A–F, normalized and averaged I_NMDA–V curves obtained with six different external Mg²⁺ concentration, showing a significant difference in voltage dependence of Mg²⁺ block properties between I_ifen-NMDA and I_NMDA. Ifen, ifenprodil.

Figure 8. The effect of voltage-dependent parameters on predicted I_NMDA–V relations for the sequential and trapping block models

A–C, predicted I_NMDA–V relations with the sequential block model showing K_Mg (0 mV), Mg²⁺ concentration and δ effects on the voltage-dependent block. A, δ, number of channels, single channel conductance, Mg²⁺ concentration and NMDA concentration were set at 0.8, 1500, 50, 1 mm and 20 μm respectively whereas in (B) and (C) δ and K_Mg (0 mV) were set at 0.8 and 1 mm respectively with the other parameters as in (A). D, simulated I_NMDA–V relations derived from the sequential model and the trapping block models. The δ and K_Mg (0 mV) were fixed to be 0.8 and 1 mm for both models, showing that under the same conditions, the trapping block model gives stronger voltage-dependent Mg²⁺ block than the sequential block model. E and F, averaged and normalized I_NMDA–V relations obtained in the absence (E) and in the presence (F) of ifenprodil were fitted using the sequential model to estimate the voltage-dependent Mg²⁺ block parameters of K_Mg (0 mV), δ and background Mg²⁺ concentration.

Figure 9. Extrasynaptic GluN2B- and GluN2D-containing NMDARs are present in postnatal day 7 substantia nigra pars compacta dopaminergic neurones

A, examples of NMDAR single channel recordings from somatic outside-out membrane patches evoked by 10 nm glutamate and 10 μm glycine. B, stability plot of channel amplitudes throughout the duration of a recording. In this recording occasional ‘double’ openings are observed indicating that more than one active channel is present in the membrane patch. C, amplitude distribution for the patch illustrated in (A), fitted with the sum of three Gaussian components. The mean amplitude and relative area of each component are shown on the histogram, and correspond in this example to conductances of 20 pS, 41 pS and 54 pS. D, stability plots of P_open, mean open time and mean shut time for the patch shown in (A). E, open time distribution for the patch illustrated in (A), fitted with a mixture of three exponential components. F, example of four types of direct transition. G, plot of channel amplitude before and after direct transitions (from the same patch shown in A). The density of points illustrates that direct transitions between 41 pS and 54 pS occur with equal frequency, while transitions between 20 pS and 41 pS are asymmetric, with 41 pS to 20 pS occurring more frequently than 20 pS to 41 pS.