CHARACTERIZATION OF THE STOICHIOMETRY OF THE NA+-BICARBONATE COTRANSPORTER

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Characterization of the stoichiometry of the Na+-bicarbonate cotransporter with self-referencing of ion-selective microelectrodes and voltage clamp. Working with bicarbonate buffered solutions is tedious due to the fact that the final bicarbonate concentration is based on the pH, temperature, ionic strength of the medium and the partial pressure of surrounding CO2. While bicarbonate can be calculated, the calculation is really only an estimate. Early work placed the stoichiometry of the Na+ /bicarbonate transporter at somewhere between 1:2 and 1:3. Later it was shown that when the transporter is in one endogenous cell line it shows a 1:2 stoichiometry and when in another endogenous cell line it shows a stoichiometry of 1:3. Our colleagues have recently expressed the transporter in a heterologous expression system to nail down the stoichiometry and determine the mechanism by which the stoichiometry can be changed. Their current setup for determining the stoichiometry of the Na+ bicarbonate has led to discrepancies. Using cells overexpressing the Na+/bicarbonate transporter, we are identifying the stoichiometry of the transporter by directly measuring H+ fluxes next to cells held under voltage clamp. By measuring the current passing through the plasma membrane along with the extracellular H+ flux we can use best fit models to determine the transporters stoichiometry and even speculate as to whether the pump is passing bicarbonate vs carbonate a more recent controversy.