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Stoichiometry and Inhibitory Pharmacology of Electrogenic Sodium Bicarbonate Cotransporter NBC4c (NBCe2‐C) Expressed in HEK‐293 Cells
Author(s) -
Shao Xuesi,
Kao Liyo,
Abuladze Natalia,
Kurtz Ira
Publication year - 2009
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.23.1_supplement.800.2
Subject(s) - dids , cotransporter , chemistry , extracellular , sodium , bicarbonate , intracellular , biophysics , stoichiometry , inhibitory postsynaptic potential , pipette , biochemistry , membrane , endocrinology , biology , organic chemistry
NBC4c is an electrogenic sodium bicarbonate cotransporter encoded by SLC4A5 . To examine the electrophysiologic properties of NBC4c in a mammalian system, HEK‐293 cells permanently expressing the cotransporter were whole‐cell patch‐clamped. When the patch pipette and bath solutions contained 0 mM HCO 3 − , increasing [HCO 3 − ]o from 0 to 25 mM induced a mean outward current of 48.4 pA at +45 mV that was blocked by DIDS and tenidap. The inhibitory profiles of DIDS and tenidap were fit to the equation I/I 0 =1/(1+(C/K i ) n ), where K i is the inhibitory constant, C is the concentration and n is the pseudo Hill coefficient. Tenidap was more potent than DIDS. Tenidap inhibition was voltage dependent with reduced inhibition at more positive voltages. At known intracellular and extracellular Na + and HCO 3 − concentrations, HCO 3 − to Na + stoichiometry (q) could be calculated with the equation: E rev = RT/(F(q‐1))ln(([Na + ] i /[Na + ] o )([HCO 3 − ] i /[HCO 3 − ] o ) q ) where reversal potential E rev (V I=0 ) was determined from the DIDS‐inhibitable I‐V curves from ‐95 mV to +45 mV. E rev was measured in two independent experiments where [HCO 3 − ] i and [HCO 3 − ] o were equal (25 mM), therefore E rev depended only on [Na + ] i /[Na + ] o . [Na + ] i /[Na + ] o values were chosen so that E rev would be significantly different when q = 2 or q = 3 predicted with the above equation. In the first protocol, using [Na + ] i /[Na + ] o = 40/80 mM, E rev = ‐22.2 ± 3.87 mV. In the second protocol using [Na + ] i /[Na + ] o = 25/135 mM, E rev = ‐40.5 ± 5.12 mV. These E rev values are close to the calculated values of ‐17.8 and ‐43.3 mV, respectively, assuming q = 2. Our data indicate that NBC4c expressed in HEK‐293 cells transports sodium bicarbonate electrogenically with a stoichiometric ratio of 2 HCO 3 − : 1 Na + . NBC4c is blocked by DIDS and tenidap with different mechanisms. Supported by NIH