Inhibition by acetazolamide of reactivated carbonic anhydrase activity in a receptor protein tyrosine phosphatase γ (RPTPγ) mutant
Authors: Duong Phu C. , Moss Fraser J. , Skelton Lara A. , Musa‐Aziz Raif , Boron Walter F. 
Published: May 2022

HCO3− reabsorption (JHCO3) by the renal proximal tubule (PT) is acutely regulated by basolateral [CO2]BL or [HCO3−]BL, but not by pHBL. RPTPγ is a transmembrane signaling protein expressed in the PT basal membrane and in many other sites throughout the body. Knocking out RPTPγ in mice renders JHCO3in isolated PTs insensitive to changes in [CO2]BL or [HCO3−]BL. Furthermore, RPTPγ knockout mice exhibit defective defense against a whole‐body metabolic acidosis. We therefore hypothesize that RPTPγ is a major CO2/HCO3− sensor in the tissues in which it is expressed. The extracellular ligand‐binding domain of RPTPγ shares 30% sequence identity to α‐type carbonic anhydrases (CAs). However, RPTPγ lacks (1) His residues at positions E149 and Q175 that participate in Zn2+ coordination in CAIV (CAIV equivalent residues, H115 and H140), and (2) the H+‐accepting His from the proton‐shuttle at the RPTPγ K122 position (CAIV residue H88). We measure extracellular surface pH (pHS) on Xenopus oocytes expressing wild‐type (WT) CAIV, RPTPg‐WT, and H2O‐injected controls. Upon switching the extracellular perfusing solution from a nominally CO2‐free ND96 solution to a 5% CO2/33mM HCO3− solution, the resulting CO2 influx causes pHS to rise, reflecting the consumption of H+ and HCO3− during the replenishment of CO2 at the cell surface. CO2 removal from the basolateral solution (“bath”) elicits the opposite effect on pHS. Consistent with prior work (Musa‐Aziz, Occhipinti & Boron. Am J Physiol, Cell Physiol 307: C814‐840, 2014), we find that CAIV increases pHS transient peak amplitude (ΔpHS) during the introduction of CO2/HCO3−. However, the ΔpHS of oocytes expressing RPTPγ‐WT is not significantly different from that measured from H2O‐injected control oocytes (n = 8 to 11; t‐test, p=0.517). We generated a mutant, in which we reconstituted 7 residues important for the catalysis (CO2 + H2O ⇌ H+ + HCO3−) by CAIV into the RPTPγ carbonic anhydrase‐like domain (CALD): T120N/K122H/A125N/E149H/Q175H/F177V/F178H (“CALD‐H”). Upon switching from ND96 (pH 7.5) to 5% CO2/33mM HCO3− (pH 7.5), the ΔpHS of oocytes expressing RPTPγ‐CALD‐H (0.330 ± 0.029; n = 12) is significantly increased vs RPTPγ‐WT (t‐test; p=3.1 × 10–7) and H2O control oocytes (t‐test; p=6.0 × 10–8). Moreover, when we add 100 mM acetazolamide (ACZ; an α‐type CA inhibitor) to the bath, the increased ΔpHS response (vs controls) of oocytes expressing either RPTPγ‐CALD‐H (0.062 ± 0.014; n = 12) or CAIV (0.077 ± 0.022; n = 7) is completely abolished (ANOVA; RPTPγ‐CALD‐H vs H2O: p=0.395; CAIV vs H2O: p=0.335). In conclusion, replacing 7 residues in RPTPγ‐CALD with their counterparts in CAIV is sufficient to re‐create CA activity. Moreover, ACZ blocks the re‐created CA activity of RPTPγ‐CALD‐H. This observation is of potential importance for the hypothesized role for WT‐RPTPγ in sensing [CO2] and [HCO3−], because it raises the possibility that sulfonamide drugs (prescribed for treating glaucoma or used as diuretics) could have the side effect inhibiting CO2/HCO3−sensing in the proximal tubule or other tissues.