Gross E, Hopfer U
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Biophys J. 1998 Aug;75(2):810-24. doi: 10.1016/S0006-3495(98)77570-8.
The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na+, three HCO3-, and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na+ and HCO3- concentrations between -160 and +160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO3- and 1 for Na+. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na+ to the cotransporter is voltage independent.
在近端小管细胞中研究了碳酸氢钠协同转运体动力学的电压依赖性。这种生电协同转运体转运一个Na⁺、三个HCO₃⁻和两个负电荷。细胞在可渗透支持物上生长至汇合，安装在Ussing型小室中，并用两性霉素B使顶端对单价小离子通透。稳态的、二硝基苯乙烯二磺酸盐敏感电流显示依赖于钠和碳酸氢盐，因此被视为通过协同转运体的通量。在零转运和对称条件下，测量了-160至+160 mV之间作为Na⁺和HCO₃⁻浓度函数的电压-电流关系。动力学可以用米氏行为来描述，HCO₃⁻的希尔系数为3，Na⁺的希尔系数为1。数据被拟合到六态有序结合模型，对限速步骤没有限制。所有有序模型都能定量解释观察到的电流-电压关系和高碳酸氢盐浓度引起的转运抑制。这些模型表明：1）空载的转运体携带正电荷；2）胞质碳酸氢盐与转运体的结合“感受”膜中12%的电场，而其跨膜转运“感受”88%的电场；3）Na⁺与协同转运体的结合与电压无关。
