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Ammonium Conductance in a Mouse Thick Ascending Limb Cell Line
Author(s) -
Lee Soojung,
Park Jonathan,
Choi Inyeong
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb752
Subject(s) - tetraethylammonium , nephron , chemistry , conductance , electrophysiology , biophysics , excretion , cotransporter , patch clamp , membrane potential , bapta , ammonium , biochemistry , intracellular , medicine , potassium , biology , sodium , receptor , mathematics , organic chemistry , combinatorics , renal function
NH 4 + is a key buffer component that regulates blood pH. The kidneys excrete NH 4 + to urine as they produce HCO 3 − , and the mechanism by which NH 4 + excretion results in net acid excretion involves a series of sophisticated NH 4 + transport processes in different parts of the nephron. One of the nephron segments that play key roles in NH 4 + 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 NH 4 + flux. Nonetheless, in vitro studies reveal that K/NH 4 exchange and NH 4 + conductance, whose molecular identities are presently unknown, can contribute to the TAL NH 4 + transport by 35–50%. The two pathways exhibit biophysical and pharmacological characteristics that distinguish them from other NH 4 + ‐transporting proteins. Despite such physiological and functional significance, our understanding of these pathways is limited. Especially, the electrophysiological properties of the NH 4 + conductance have been unknown. In this study, we examined the NH 4 + conductance in the TAL cell line ST‐1 to determine its basic electrophysiological properties such as the amounts of NH 4 + 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 NH 4 Cl in the presence of BaCl 2 , tetraethylammonium and BAPTA. Application of 20 mM NH 4 Cl induced an inward current with mean amplitude of −272 ± 79 pA (n = 9). In I–V relationships, NH 4 Cl application caused the I–V curve to shift down in an inward direction. The difference in curves before and after NH 4 Cl application, which corresponds to the NH 4 Cl‐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 NH 4 Cl in the presence of BaCl 2 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 NH 4 + conductance was still produced while endogenous oocyte conductance was not detected. In conclusion, we, for the first time, report the electrophysiological properties of the NH 4 + conductance in the ST‐1 cells. This conductance is Na + ‐dependent, different from the previously reported Cl − ‐dependent NH 4 + conductance in isolated TAL tubules. Support or Funding Information This work was supported by the Emory physiology department bridge fund to I.C.