Evidence for ammonium conductance in a mouse thick ascending limb cell line.

Soojung Lee,Inyeong Choi,Jun Ming Li,Kathy Li,Jonathan Park

doi:10.14814/phy2.13379

Abstract

In this study, we examined an ammonium conductance in the mouse thick ascending limb cell line ST‐1. Whole cell patch clamp was performed to measure currents evoked by NH 4Cl in the presence of BaCl2, tetraethylammonium, and BAPTA. Application of 20 mmol/L NH 4Cl induced an inward current (−272 ± 79 pA, n = 9). In current‐voltage (I–V) relationships, NH 4Cl application caused the I–V curve to shift down in an inward direction. The difference in current before and after NH 4Cl application, which corresponds to the current evoked by NH 4Cl, was progressively larger at more negative potentials. The reversal potential for NH4Cl was +15 mV, higher than the equilibrium potential for chloride, indicating that the current should be due to NH 4 +. We then injected ST‐1 poly(A) RNA into Xenopus oocytes and performed two‐electrode voltage clamp. NH 4Cl application in the presence of BaCl2 caused the I–V curve to be steeper. The NH 4 + current was retained at pH 6.4, where endogenous oocyte current was abolished. The NH 4 + current was unaffected by 10 μmol/L amiloride but abolished after incubation in Na+‐free media. These results demonstrate that the renal cell line ST‐1 produces an NH 4 + conductance.

Highlights

NHþ4 is a key buffer component that regulates blood pH
These results demonstrate that the renal cell line ST-1 produces an NH4+ conductance
We examined an NHþ4 conductance in the mouse thick ascending limb (TAL) cell line ST-1

Summary

Introduction

NHþ4 is a key buffer component that regulates blood pH. In essence, the kidneys excrete NHþ4 to urine as they produce HCOÀ3 , and the mechanism by which NHþ4 excretion results in net acid excretion involves a series of sophisticated NHþ4 transport processes in different parts of the nephron (Weiner and Verlander 2013; Hamm et al.2015). One of the nephron segments that play key roles in NHþ4 excretion is the thick ascending limb (TAL) (Mount 2014). NHþ4 transport in the TAL involves the Na/K/2Cl cotransporter NKCC2 (Good et al 1984; Kinne et al 1986), K/NH4 exchange and NHþ4 conductance (Amlal et al 1994; Attmane-Elakeb et al 2001) in the luminal membrane of the tubule, and the Na/H exchanger. In the luminal membrane of the TAL, NKCC2 is the major fraction of the active NHþ4 flux. In vitro studies reveal that K/NH4 exchange and NHþ4 conductance can contribute to the NHþ4 transport by 35–50% (Amlal et al 1994; Attmane-Elakeb et al 2001)

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