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

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 NH 4 + and NH 3 . 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 CO 2 transport by AQP1. This suggested that DIDS might also inhibit transport of other gases such as NH 3 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 (pH s ) and whole cell currents (I) induced by NH 3 /NH 4 + . All experiments were conducted on Xenopus oocytes expressing Rhbg. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH 4 Cl caused a decrease in pH s by 0.85±0.12 and an inward current of ‐58±10 nA. The decrease in pH s is caused by NH 3 influx whereas the inward current is due to electrogenic NH 4 + influx. In the presence of DIDS, NH 4 Cl caused a significantly smaller decrease in pH s (0.57±0.07) and current (‐29±5 nA). Similarly, exposing Rhbg‐oocytes to 5mM MA/MA + (a substitute to NH 3 /NH 4 + ) caused a decrease in pH s of 0.27±0.04 and an inward current of ‐29±6 nA. In the presence of DIDS, the MA/MA + induced changes in pH s (0.17±0.03) and ΔI (‐11±2 nA) were also inhibited. DIDS had no effect on NH 3 /NH 4 + transport in H 2 O‐injected oocytes. Our data indicate that 1) DIDS inhibits transport of NH 3 & MA by Rhbg without affecting endogenous NH 3 & MA transport. 2) DIDS also inhibits the electrogenic transport of NH 4 + & MA + by Rhbg. 3) This is the first report to show inhibition of both gas (NH 3 ) and ionic (NH 4 + ) transport by Rhbg. Grant Funding Source : Supported by VA Merit grant