Ammonium Conductance in a Mouse Thick Ascending Limb Cell Line

Abstract

NH4+ is a key buffer component that regulates blood pH. The kidneys excrete NH4+ to urine as they produce HCO3−, and the mechanism by which NH4+ excretion results in net acid excretion involves a series of sophisticated NH4+ transport processes in different parts of the nephron. One of the nephron segments that play key roles in NH4+ excretion is the thick ascending limb (TAL). In the luminal membrane of the TAL tubules, the Na/K/2Cl cotransporter is the major fraction of the active NH4+ flux. Nonetheless, in vitro studies reveal that K/NH4 exchange and NH4+ conductance, whose molecular identities are presently unknown, can contribute to the TAL NH4+ transport by 35–50%. The two pathways exhibit biophysical and pharmacological characteristics that distinguish them from other NH4+‐transporting proteins. Despite such physiological and functional significance, our understanding of these pathways is limited. Especially, the electrophysiological properties of the NH4+ conductance have been unknown.In this study, we examined the NH4+conductance in the TAL cell line ST‐1 to determine its basic electrophysiological properties such as the amounts of NH4+current produced in cells, current‐voltage (I–V) relationship, and reversal potential of the current. Whole cell patch clamp was performed to measure the current evoked by NH4Cl in the presence of BaCl2, tetraethylammonium and BAPTA. Application of 20 mM NH4Cl induced an inward current with mean amplitude of −272 ± 79 pA (n = 9). In I–V relationships, NH4Cl application caused the I–V curve to shift down in an inward direction. The difference in curves before and after NH4Cl application, which corresponds to the NH4Cl‐mediated currents at different voltages, was progressively larger at more negative potentials. The reversal potential was +15 mV, more positive than a calculated equilibrium potential for Cl−, thus indicating that the current is not due to Cl−but to NH +. We then expressed ST‐1 total proteins in Xenopus oocytes and performed two‐electrode voltage clamp. Application of NH4Cl in the presence of BaCl2 caused the I–V curve to be steeper in an inward direction. Interestingly, removing Na+ from the bath solution caused the NH4+ conductance to disappear. Na+removal additionally led to a partial reduction of endogenous oocyte conductance, which was abolished by bumetanide. At pH 6.4, the NH4+conductance was still produced while endogenous oocyte conductance was not detected. In conclusion, we, for the first time, report the electrophysiological properties of the NH4+conductance in the ST‐1 cells. This conductance is Na+‐dependent, different from the previously reported Cl−‐dependent NH4+conductance in isolated TAL tubules.Support or Funding InformationThis work was supported by the Emory physiology department bridge fund to I.C.