Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Abstract

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ & RPTPζ) are transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPγ and RPTPζ are unusual in having an extracellular carbonic-anhydrase–like domain (CALD). Carbonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ⇌ HCO3– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃⁻ sensor. RPTPγ—expressed on the PT basal (i.e., blood-side) membrane—is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃⁻]. RPTPγ and RPTPζ are also present in the central nervous system (CNS), including mouse hippocampus (HC). RPTPγ is expressed primarily in neurons and RPTPζ, in astrocytes. Here, we investigate whether RPTPγ and RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]ₒ and [HCO₃⁻]ₒ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPs regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃⁻]ₒ) or respiratory acidosis (RAc, caused by ↑ [CO₂]ₒ). We propose that the RPTPs respond to ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ by promoting a shift from dimers to monomers, thereby dis-inhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTP dis-inhibition would be increased acid-extrusion rate (JE) or decreasedacid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a primary antibody against the RPTPγ fibronectin III domain shows that RPTPγ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes . RT-PCR cloning from mixed HC cultures identifies specific RPTPγ and RPTPζ variants. Presumably it is the neurons that express two identified RPTPγ variants: the full-length protein, encoded by all 30 ptprg exons (NM_008981), and a second newly-detected variant that corresponds to the hypothetical assembly X1 (XM_006517956). The HC co-cultures also express six RPTPζ variants: Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). To assess the influence of RPTPζ on pHi homeostasis, we loaded cells with the pH-sensitive dye BCECF, digitally monitored dye fluorescence, and computed pHi of cells subjected to acid-base disturbances. In MAc-MAc protocols, the effects of the knockout (KO) of RPTPζ are unremarkable in both neurons and astrocytes. In RAc-RAc protocols, the KO has unremarkable effects on neurons. However, in astrocytes the RPTPζ-KO markedly increases both the rates and the magnitudes of pHi decrease during both RAc pulses. Thus, RPTPζ may stabilize pHi in astrocytes subjected to RAc.