Choi I et al. (2022), Bicarbonate-Independent Sodium Conductance of N...

XB-ART-59167

Curr Issues Mol Biol 2022 Mar 13;443:1284-1293. doi: 10.3390/cimb44030086.

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Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function.

Choi I , Yang H , Kim E , Lee S .

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The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na+ in a HCO3--independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na+ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO2/HCO3-, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current-voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO2/HCO3-. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na+ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.

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Genes referenced: dlg4 slc4a7

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	Figure 1. NBCn1 channel activity. (A) I–V relationships in oocytes expressing NBCn1 or water-injected control oocytes. Oocytes were subjected to step-voltage commands from –120 to +60 mV (20 mV steps) in CO2/HCO3−-free ND96 solution (n = 5–7/group). The increased slope of the I–V plot is a hallmark for NBCn1 channel activity. (B) INBCn1–V relationship of NBCn1 channel activity. The plot was obtained from the difference between the I–V plots in the presence and absence of bath Na+. (C) Resting membrane potentials (Vm). Vm was measured 48–72 h after cRNA injection (n = 5 controls and 16 NBCn1). ** p < 0.01, Student t-test.
	Figure 2. NBCn1 channel activity decreases IGlu produced by GluN1·N2. (A) Representative IGlu produced by GluN1·N2. IGlu was measured with an application of 100 µM glutamate (10 µM glycine, no Mg2+). Recording was performed in CO2/HCO3−-free ND96 solution containing 96 mM NaCl (holding potential of –40 mV). (B–D) Representative IGlu produced by GluN1·N2/NBCn1. A fixed amount of GluN1·N2 was coexpressed with 1, 5, or 10 ng of NBCn1. (E) Control. (F) Mean IGlu. Data were obtained from 4–5 oocytes/group. * p < 0.05, ** p < 0.01 compared to GluN1·N2 alone, one-way ANOVA with Dunnett post-test. (G) and (H) I–V relationships of glutamate-evoked responses by GluN1·N2 (n = 6; (G)) and GluN1·N2/NBCn1 (n = 5; (H)). (I) IGlu–V relationships. IGlu is the mean difference before and after glutamate application in (G) and (H). NBCn1 alone served as a control (n = 4).
	Figure 3. PSD95 increases IGlu produced by GluN1·N2. (A) I–V relationships of glutamate-evoked responses by GluN1·N2 (n = 3). (B) I–V relationships of glutamate-evoked responses by GluN1·N2/PSD95 (n = 5). (C) IGlu–V relationships. IGlu is the mean difference before and after glutamate application in (A) and (B).
	Figure 4. PSD95 has negligible effect on IGlu in the presence of NBCn1. (A) I–V relationships of glutamate-evoked responses by GluN1·N2/NBCn1 (n = 11). (B) I–V relationships of glutamate-evoked responses by GluN1·N2/PSD95/NBCn1 (n = 7). (C) IGlu–V relationships. IGlu is the mean difference before and after glutamate application in (A,B).
	Figure 5. The IGlu decrease by NBCn1 channel activity is independent of CO2/HCO3−. (A,B) Representative IGlu produced by GluN1·N2/PSD95 (A) and GluN1·N2/PSD95/NBCn1 (B). IGlu was measured in ND96 solution and 10 min after applying a solution equilibrated with 10% CO2, 50 mM HCO3− at constant pH 7.4. Except when glutamate was applied, Na+-free CO2/HCO3− solution was applied to minimize pHi change by cotransport activity. (C) Mean IGlu produced by GluN1·N2/PSD95 (n = 4; (C)) and GluN1·N2/PSD95/NBCn1 (n = 6; (D)). (E) Effects of NBCn1 on IGlu in the absence and presence of CO2/HCO3−. Data were presented as percent change relative to IGlu by GluN1·N2/PSD95. ** p < 0.01.

References [+] :

Aalkjaer, Cation-coupled bicarbonate transporters. 2014, Pubmed