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.
Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport
Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-4951; E-mail: [email protected]
Portions of this work have been previously published in preliminary form (Bevensee, M.O., W.F. Boron, and M. Apkon. 1995. FASEB J. 9: A308.).
Abbreviations used in this paper: BCECF, 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein; DIA, depolarization-induced alkalinization; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; MQAE, N-(6-methoxyquinolyl)acetoethyl ester; Vm, plasma membrane voltage.
The sum of pHi increases in Fig. 5, ab, cd, and ef was 0.51. βT is the sum of βI (12.7 mM [pH unit]−1) and βHCO3− (12.1 mM [pH unit]−1), or 24.8 mM (pH unit)−1 at an average pHi of 6.79. 0.51 pH units × 24.8 mM (pH unit)−1 × 0.75 (DIDS-sensitive fraction, as described in the accompanying paper; Bevensee et al., 1997) = 9.5 mM HCO3− moved into the cell by the Na-driven HCO3− transporter. For a Na-driven Cl-HCO3 exchanger, two HCO3− ions would move into the cell in exchange for one Cl− ion out of the cell. Therefore, the movement of 9.5 mM HCO3− into the cell by this exchanger would result in the movement of 4.7 mM Cl− out of the cell.
Removing external Na+ in CO2/HCO3− elicited a HCO3− efflux of 101 μM s−1, of which 90% or 91 μM s−1 was DIDS sensitive. This DIDS-sensitive HCO3− efflux corresponds to the net movement of 4.55 C (liter cell vol)−1 s−1 for a Na/HCO3 cotransporter that moves one charge equivalent per two HCO3− ions. We calculated the average cell volume of an astrocyte to be 1.6 pl, assuming the geometry of an astrocyte to be a right-angled cone (V = [area × h]/3) with a height (h) of 5 μm (O'Connor et al., 1993). We imaged BCECF-loaded astrocytes to obtain an average cell area of 973 ± 70 μm2 (n = 17). Therefore, the current flow through the Na/HCO3 cotransporter in a typical astrocyte would be 7.3 pA. Given an average input resistance for hippocampal astrocytes of 236 MΩ (see below), this 7.3-pA current would depolarize astrocytes by 1.7 mV.
We sometimes observed such abrupt depolarizations in experiments in which we monitored only Vm, as in Fig. 10,A. Conversely, we sometimes observed slower depolarizations in experiments in which we monitored Vm and pHi simultaneously. One would expect such depolarizations due to electrogenic Na/HCO3 cotransport to be instantaneous under conditions in which the bath solution could be instantaneously changed. However, to maintain seals in our patch-clamp experiments performed at 37°C, we were often forced to reduce our solution flow rate. During such experiments, we often observed the slower depolarizations in response to Na+ removal (e.g., Fig. 10 A).
35.8 μM s−1 corresponds to the net movement of 1.79 C (liter cell vol)−1 s−1 for a Na/HCO3 cotransporter that moves one charge equivalent per two HCO3− ions. The current flow through the Na/HCO3 cotransporter in a typical astrocyte with a volume of 1.6 pl would be 2.9 pA. This current flow would depolarize hippocampal astrocytes with an input resistance of 236 MΩ by only 0.7 mV.
Mark O. Bevensee, Michael Apkon, Walter F. Boron; Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport. J Gen Physiol 1 October 1997; 110 (4): 467–483. doi: https://doi.org/10.1085/jgp.110.4.467
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