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II. Electrogenic Na/HCO3 Cotransport
,* Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520
In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na+-driven influx of HCO3– causes the increase in intracellular pH (pHi) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO2/17 mM HCO3–. In the present study, we used the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na+-driven Cl-HCO3 exchanger, an electrogenic Na/HCO3 cotransporter, or an electroneutral Na/HCO3 cotransporter. To determine if the transporter is a Na+-driven Cl-HCO3 exchanger, we depleted the cells of intracellular Cl– by incubating them in a Cl–-free solution for an average of
11 min. We verified the depletion with the Cl–-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl–-depleted cells, the pHi still increases after one or more exposures to CO2/HCO3–. Furthermore, the pHi decrease elicited by external Na+ removal does not require external Cl–. Therefore, the transporter cannot be a Na+-driven Cl-HCO3 exchanger. To determine if the transporter is an electrogenic Na/ HCO3 cotransporter, we measured pHi and plasma membrane voltage (Vm) while removing external Na+, in the presence/absence of CO2/HCO3– and in the presence/absence of 400 µM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). The CO2/HCO3– solutions contained 20% CO2 and 68 mM HCO3–, pH 7.3, to maximize the HCO3– flux. In pHi experiments, removing external Na+ in the presence of CO2/HCO3– elicited an equivalent HCO3– efflux of 281 µM s–1. The HCO3– influx elicited by returning external Na+ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive Vm change was 3.3 mV. Indeed, we found that removing external Na+ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO2/ HCO3–. Thus, the Na/HCO3 cotransporter is electrogenic. Because a cotransporter with a Na+:HCO3– stoichiometry of 1:3 or higher would predict a net HCO3– efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO3 cotransporter with a stoichiometry of 1:2.
Key Words: acid–base transport • glia • intracellular Cl– • N -(6-methoxyquinolyl)acetoethyl ester • patch clamp
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