Rhbg interaction with CA-IV and its effects on NH3/NH4+ and CO2 transport.

Renal Rhesus type B glycoprotein (Rhbg) is a glycosylated mammalian NH3/NH4+ transporter expressed in α-intercalated cells of the collecting duct. Carbonic anhydrase-IV (CA-IV) is also expressed in the mammalian kidney, where it catalyzes the reversible hydration of CO2. This study aims to demonstrate: 1) whether Rhbg and CA-IV proteins physically interact; and 2) if this interaction functionally affects transport of NH3/NH4+ and possibly CO2. We measured transport of NH4+, NH3, and CO2 in four groups of Xenopus oocytes. In the first group, we coexpressed Rhbg with CA-IV and compared the measurements to three groups of oocytes expressing either Rhbg or CA-IV or injected with H2O. We used ion-selective microelectrodes to measure surface pH, to monitor NH3 transport, and intracellular pH to monitor NH4+ and CO2 transport. We also used a two-electrode voltage clamp to measure current changes caused by electrogenic NH4+ transport. These parameters measured NH3/NH4+ and CO2 transport in oocytes expressing Rhbg and/or CA. Our results indicate that: 1) Rhbg and CA-IV were coimmunoprecipitated, suggesting a physical interaction; and 2) coexpressing CA-IV with Rhbg: i) inhibited electrogenic NH4+ transport by Rhbg in the presence and absence of CO2; ii) reduced NH3 transport by Rhbg only in the presence of CO2; and iii) had no detectable effect on CO2 transport by Rhbg. We demonstrated for the first time that Rhbg and CA-IV physically interact, and this interaction has inhibitory effects on Rhbg function but not CA-IV. The interaction of Rhbg and CA-IV is important to explain their role in renal acid-base homeostasis.NEW & NOTEWORTHY Our study revealed the complex regulation of NH3/NH4+ transport, highlighting the roles of Rhbg, CA-IV, and environmental factors such as CO2 concentration. These interactions are critical to our understanding of NH3/NH4+ transport and regulation. Our findings lay a strong foundation for future investigations into the molecular dynamics among these transport proteins and their physiological significance. These studies are essential to fully understand how these mechanisms influence renal ammonia handling, urinary acidification, and systemic pH balance.