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

Abstract

Renal Rhesus type B glycoprotein (Rhbg) is a glycosylated mammalian NH3/NH4+ transporter expressed in a-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.