The Effects of Co‐Expression of Rhbg with Human CA‐IV on NH 3 /NH 4 + and CO 2 Transport

Abstract

Background Rhbg is a member of the mammalian glycosylated rhesus (Rh) protein family expressed in multiple tissues including the basolateral membrane of the renal collecting duct. Rhbg was found to transport NH4+ and NH3 in the collecting duct. Carbonic anhydrase IV (CA-IV) is one of several carbonic anhydrases expressed in the mammalian kidney where it catalyzes the reversible hydration of CO2. It is anchored at both apical and basolateral membranes of renal proximal tubules and the collecting duct. Objective and Hypothesis Our previous studies showed that Rhbg and CA-IV proteins co-immunoprecipitated, suggesting that there is a physical interaction between Rhbg and CA-IV. This study is to detect if there is any functional effect related to this interaction. Methods We measured transport of NH4+, NH3, and CO2 in 4 groups of Xenopus oocytes expressing the following: In the first group, we co-expressed the mouse Rhbg with human CA-IV (by injecting oocytes with Rhbg and CA-IV mRNA) and compared the measurements to 3 groups of oocytes expressing either Rhbg or CA-IV, or injected with H2O. We used ion-selective microelectrodes to measure changes in surface pH (pHs), to monitor NH3 transport, and changes in intracellular pH (pHi) to monitor NH4+ and CO2 transport. We also used two-electrode voltage-clamp to measure current change (ΔI) caused by electrogenic NH4+ transport. In each oocyte, we monitored one of the three parameters indicated above (pHs, pHi or ΔI), in response to five experimental conditions: 1) increased CO2 (1.5%CO2/10mM HCO3-); 2) NH3/NH4+ (5mM NH4Cl in HEPES); 3) Methylamine/Methylammonium, often used as a substitute for ammonium (MA/MA+, 5mM in HEPES); 4) 5mM NH3/NH4+ in 1.5%CO2; 5) 5mM MA/MA+ in 1.5% CO2. Results Our results indicate that co-expressing CA-IV with Rhbg: 1) inhibited electrogenic NH4+ transport by Rhbg in the presence and absence of CO2; 2) reduced MA and NH3 transport by Rhbg only in the presence of CO2; 3) had no detectable effect on CO2 transport by Rhbg. Conclusions We demonstrated for the first time that co-expression of Rhbg and CA-IV inhibited electrogenic transport of NH4+ by Rhbg and reduced NH3 and MA transport in the presence of CO2. The function of Rhbg as a CO2 gas channel was demonstrated as an increase in the rate of CO2-induced pHiacidification in Rhbg expressing oocytes and was not altered by expression of CA-IV. The interaction of Rhbg and CA-IV is important to explain their role in renal acid-base homeostasis. We propose that NH3/NH4+ transport by Rhbg is modified in the presence of CO2.