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: <i>1</i>) whether Rhbg and CA-IV proteins physically interact; and <i>2</i>) if this interaction functionally affects transport of NH₃/NH₄⁺ and possibly CO₂. We measured transport of NH₄⁺, NH₃, and CO₂ in four groups of <i>Xenopus</i> 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: <i>1</i>) Rhbg and CA-IV were coimmunoprecipitated, suggesting a physical interaction; and <i>2</i>) coexpressing CA-IV with Rhbg: <i>i</i>) inhibited electrogenic NH₄⁺ transport by Rhbg in the presence and absence of CO₂; <i>ii</i>) reduced NH₃ transport by Rhbg only in the presence of CO₂; and <i>iii</i>) 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.<b>NEW & NOTEWORTHY</b> 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.