Inactivation of a volume-sensitive basolateral potassium conductance in turtle colon: effect of metabolic inhibitors

The Journal of Physiology

Abuladze

Pushkin

et al. 2001

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3−:Na+ stoichiometry is not larger then 2:1. However, there are no data available on either the reversal potential or the HCO3−:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT‐PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current‐voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current‐voltage relationships, all corresponded to a 2 HCO3−:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3−:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Section: Resultsmentioning

confidence: 99%

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO₃⁻:1 Na⁺

The Journal of Physiology

Abuladze

Pushkin

et al. 2001

“…Functional studies were performed 48 h after transfection. The cells were permeabilized apically with 10 M amphotericin B to remove the electrical resistance of the apical membrane (4,7,(16)(17)(18). The application of a Na ϩ gradient across the epithelial cell monolayer in cells expressing NBC4c in the presence of HCO 3 Ϫ generates two currents with opposite signs.…”

Section: Methodsmentioning

confidence: 99%

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter

Sassani

Pushkin

American Journal of Physiology-Cell Physiology

et al. 2002

114

Sodium-bicarbonate cotransporters are homologous membrane proteins mediating the electrogenic or electroneutral transport of sodium and bicarbonate. Of the functionally characterized sodium-bicarbonate cotransporters (NBC), NBC1proteins are known to be electrogenic. Here we report the cloning and functional characterization of NBC4c, a new splice variant of the NBC4 gene. At the amino acid level, NBC4c is 56% identical to NBC1 protein variants and 40% identical to electroneutral NBC3. When expressed in mammalian cells, NBC4c mediates electrogenic sodium-bicarbonate cotransport. The transport of sodium and bicarbonate is chloride independent and is completely inhibited by DIDS. NBC4c transcripts were detected in several tissues including brain, heart, kidney, testis, pancreas, muscle, and peripheral blood leukocytes. The data indicate that NBC4c is an electrogenic sodium-bicarbonate cotransporter. The finding that both NBC1 and NBC4c proteins function as electrogenic sodium-bicarbonate cotransporters will aid in determining the structural motifs responsible for this unique functional property, which distinguishes these transporters from other members of the bicarbonate transporter superfamily.

“…Permeabilization of the apical membrane with amphotericin B "removes" the electrical resistance of that membrane and reveals the basolateral electrogenic processes to external electrodes. The use of amphotericin B for the above-mentioned purposes is a common practice in studies of epithelial transport (Kirk and Dawson, 1983;Backman et al, 1992;Illek et al, 1993;Acevedo, 1994;Gross and Hopfer, 1996). In a previous study, we found that the current generated by Na,K-ATPase under short-circuit conditions interferes with measurements of currents related to Na-HCO 3 cotransporter activity (Gross and Hopfer, 1996).…”

Section: Experimental Strategymentioning

confidence: 96%

Voltage and Cosubstrate Dependence of the Na-HCO3 Cotransporter Kinetics in Renal Proximal Tubule Cells

Hopfer

1998

Biophysical Journal

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.