Glial Nax channels control lactate signaling to neurons for brain [Na+] sensing

Abstract

Sodium (Na) homeostasis is crucial for life and Na levels in body fluids are constantly monitored in the brain. The subfornical organ (SFO) is the center of the sensing responsible for the control of Na-intake behavior, where Na x channels are expressed in specific glial cells as the Na-level sensor. Na x channel is a concentration-sensitive Na channel with a threshold value of approximately 150 mM for the extracellular Na ion.The Na x -positive glial cells are sensitive to an increase in the extracellular Na level in the physiological range, indicating that glial cells, not neurons, are the primary site of Na-level sensing. However, the mechanism by which the Na signal sensed by "inexcitable" glial cells is transferred to neurons has remained to be elucidated.To gain insight into the cellular processes involving Na x in glial cells, in this doctor thesis, I started in my study with screening for molecules interacting with Na x using the yeast two-hybrid system with each of the cytoplasmic domains of mouse Na x as bait. Among the positive clones isolated from a mouse DRG cDNA library by using the C-terminal region of Na x as bait, three clones coded for the α subunit of Coexpression of the α1 subunit of Na + /K + -ATPase and Na x channels was examined by double-fluorescent immunostaining using sections of the SFO and dissociated cells from the SFO. The α1 subunit was broadly distributed throughout the iii SFO, overlapping with the expression of Na x channels. The confocal microscopic analyses with isolated cells from the SFO showed that both molecules were colocalized in the plasma membrane. Na x channels were expressed in large round cells, but not in small cells with neurite-like processes, indicating that Na x channels are expressed in glial cells including ependymal cells.It is known that the α2 and α3 isoforms of Na + /K + -ATPase are also expressed in the brain. Experiments using the yeast two-hybrid system showed that the cytoplasmic fragment of α2 corresponding to the region of the α1 isoform isolated also interacted with the C-terminal region of Na x , but that of α3 did not. Thus, Na x has specific interaction with α1 and α2 isoforms of Na and α2 subunits significantly suppressed the metabolic response in the C6M16 cells with Na x expression in the 170 mM Na solution. In contrast, overexpression of the fragment of the α3 subunit, which was negative for interaction with Na x channel, did not affect the metabolic activation.The C-terminal fragment of Na x was also expected to work as a competitor for the binding of Na x channels to Na + /K + -ATPase. Unexpectedly but intriguingly, overexpression of the C-terminal fragment of Na x further enhanced the 2-NBDG uptake, when it was coexpressed in Na x -positive cells. This suggests that the C-terminal region of Na x is also able to support Na + /K + -ATPase, as well as the native Na x . However, the expression of the C-terminal fragment of Na x by itself (without concomitant expression of the native Na x ) exerted no effect on the 2-NBDG uptake. This ...

SAP97 promotes the stability of Nax channels at the plasma membrane

“…We previously established a C6 glioblastoma cell line, C6M16, in which the expression of Na x channels is inducible under the control of the TRE [11]. For the present study, we established another C6 cell line, C6M(TA)8 expressing the Na x (T1679A) mutant with a similar expression level of Na x proteins to C6M16 cells, to characterize differences from the wild type.…”

“…The Glutathione S-transferase (GST)-fused protein with the C-terminal region (amino acid residues 1489-1681) of mouse Na x (GST-Na x -C-term), its PDZ-binding-motif mutant (GST-Na x -Cterm-T1679A) in which Thr-1679 was replaced with Ala, or its PDZ-binding-motif deletion mutant (GST-Na x -C-termDTQI) was expressed in an Escherichia coli strain, BL21, and purified by glutathione affinity chromatography as described [11].…”

“…The expression construct of the PDZ-binding motif mutant (pTRE-mNa x -T1679A), in which Thr-1679 was replaced with Ala, was generated from pTRE-mNa x [11] using the QuickChange Mutagenesis kit (Stratagene). Expression plasmids for the N-terminal FLAG-tagged wild-type Na x (pFLAG-mNa x ) and mutant Na x (pFLAG-mNa x -T1679A) were generated by subcloning the respective cDNAs, into pcDNA-FLAG [14].…”

“…The C6M16 cell line, in which mouse Na x expression is inducible under the control of the tetracycline-responsive element (TRE), was described previously [11]. Cell lines which allow inducible expression of a PDZ-binding-motif mutant of Na x were established in the same way.…”

Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells

“…To date, apart from the related Nax which appears to function as a sodium sensor [1,2], nine isoforms termed Nav1.1 -Nav1.9 have been functionally defined as sodium-selective ion channels [3]. Their distinct tissue distribution and amenability to modulation by toxins and drugs has led to significant interest in Nav as therapeutic targets in a number of poorly treated conditions ranging from epilepsy to cardiac arrhythmias and pain [4].…”