Inhibition by acetazolamide of reactivated carbonic anhydrase activity in a receptor protein tyrosine phosphatase γ (RPTPγ) mutant.

HCO reabsorption (J ) by the renal proximal tubule (PT) is acutely regulated by basolateral [CO ] or [HCO ] , but not by pH . 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 J in isolated PTs insensitive to changes in [CO ] or [HCO ] . Furthermore, RPTPγ knockout mice exhibit defective defense against a whole-body metabolic acidosis. We therefore hypothesize that RPTPγ is a major CO /HCO 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 Zn 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 (pH ) on Xenopus oocytes expressing wild-type (WT) CAIV, RPTPg-WT, and H O-injected controls. Upon switching the extracellular perfusing solution from a nominally CO -free ND96 solution to a 5% CO /33mM HCO solution, the resulting CO influx causes pH to rise, reflecting the consumption of H and HCO during the replenishment of CO at the cell surface. CO removal from the basolateral solution ("bath") elicits the opposite effect on pH . Consistent with prior work (Musa-Aziz, Occhipinti & Boron. Am J Physiol, Cell Physiol 307: C814-840, 2014), we find that CAIV increases pH transient peak amplitude (ΔpH ) during the introduction of CO /HCO . However, the ΔpH of oocytes expressing RPTPγ-WT is not significantly different from that measured from H O-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 (CO + H O ⇌ H + HCO ) 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% CO /33mM HCO (pH 7.5), the ΔpH of oocytes expressing RPTPγ-CALD-H (0.330 ± 0.029; n = 12) is significantly increased vs RPTPγ-WT (t-test; p=3.1 × 10 ) and H O control oocytes (t-test; p=6.0 × 10 ). Moreover, when we add 100 mM acetazolamide (ACZ; an α-type CA inhibitor) to the bath, the increased ΔpH 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 H O: p=0.395; CAIV vs H O: 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 [CO ] and [HCO ], because it raises the possibility that sulfonamide drugs (prescribed for treating glaucoma or used as diuretics) could have the side effect inhibiting CO /HCO sensing in the proximal tubule or other tissues.