Inhibition of Rhbg‐mediated NH3 and NH4+ transport by DIDS (1098.2)

Abstract

Rhbg is a non‐erythroid Rh glycoprotein expressed at the basolateral membrane of renal collecting duct. In previous studies, we demonstrated that Rhbg transported both NH4+ and NH3. As such, the Rh transporter is unique in being able to transport both the ionic and the gaseous components of ammonia. Other studies have shown that DIDS inhibited CO2 transport by AQP1. This suggested that DIDS might also inhibit transport of other gases such as NH3 by Rh proteins. This study was conducted to test this hypothesis. To do so we used ion‐selective microelectrodes and two‐electrode voltage clamp to measure changes in surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+. All experiments were conducted on Xenopus oocytes expressing Rhbg. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH4Cl caused a decrease in pHs by 0.85±0.12 and an inward current of ‐58±10 nA. The decrease in pHs is caused by NH3 influx whereas the inward current is due to electrogenic NH4+ influx. In the presence of DIDS, NH4Cl caused a significantly smaller decrease in pHs (0.57±0.07) and current (‐29±5 nA). Similarly, exposing Rhbg‐oocytes to 5mM MA/MA+ (a substitute to NH3/NH4+) caused a decrease in pHs of 0.27±0.04 and an inward current of ‐29±6 nA. In the presence of DIDS, the MA/MA+ induced changes in pHs (0.17±0.03) and ΔI (‐11±2 nA) were also inhibited. DIDS had no effect on NH3/NH4+ transport in H2O‐injected oocytes.Our data indicate that 1) DIDS inhibits transport of NH3 & MA by Rhbg without affecting endogenous NH3 & MA transport. 2) DIDS also inhibits the electrogenic transport of NH4+ & MA+ by Rhbg. 3) This is the first report to show inhibition of both gas (NH3) and ionic (NH4+) transport by Rhbg.Grant Funding Source: Supported by VA Merit grant