Rhbg Interaction with CA-IV and its effects on NH

Background: Renal Rhbg is a glycosylated mammalian NH3/NH4+ transporter expressed in alpha-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. Methods: We measured transport of NH4+, NH3, and CO2 in 4 groups of Xenopus oocytes. In the first group, we co-expressed Rhbg with CA-IV 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 surface pH, to monitor NH3 transport, and intracellular pH to monitor NH4+and CO2 transport. We also used 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. Results: Our results indicate that: 1) Rhbg and CA-IV were co-immunoprecipitated, suggesting a physical interaction; and 2) co-expressing 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; iii) had no detectable effect on CO2 transport by Rhbg. Conclusions: 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.